Efficient optical analysis of polymers using arrays of nanostructures

ABSTRACT

The invention is directed to methods and apparatus for detecting sequences of optical signals from parallel reactions on an array of nanostructures, such as nanopores, nanowells, or nanoparticles. In accordance with the invention, an array of nanostructures is provided, each nanostructure comprising a reaction site and each capable of confining a reaction that generates a sequence of optical signals, and the nanostructures of the array being arranged in clusters each comprising a number of nanostructures. Each different cluster is disposed within a different resolution limited area and the number of nanostructures in each cluster is either greater than one or a random variable with an average value greater than zero. Optical signals from reactions in the nanostructures are detected by an optical system operatively associated with the array.

This application claims priority from U.S. provisional application Ser.No. 62/068,599 filed 24 Oct. 2014, which is hereby incorporated byreference in its entirety.

BACKGROUND

DNA sequencing technologies developed over the last decade haverevolutionized the biological sciences, e.g. Lerner et al, The Auk, 127:4-15 (2010); Metzker, Nature Review Genetics, 11: 31-46 (2010); Holt etal, Genome Research, 18: 839-846 (2008); and have the potential torevolutionize many aspects of medical practice in coming years, e.g.Voelkerding et al, Clinical Chemistry, 55: 641-658 (2009); Anderson etal, Genes, 1: 38-69 (2010); Freeman et al, Genome Research, 19:1817-1824 (2009); Tucker et al, Am. J. Human Genet., 85: 142-154 (2009).However, to realize such potential there are still a host of challengesthat must be addressed, including reduction of per-run sequencing cost,simplification of sample preparation, reduction of run time, increasingsequence read lengths, improving data analysis, and the like, e.g.Baker, Nature Methods, 7: 495-498 (2010); Kircher et al, Bioessays, 32:524-536 (2010); Turner et al, Annual Review of Genomics and HumanGenetics, 10: 263-284 (2009). Single molecule sequencing onnano-fabricated arrays, such as nanopore arrays, may address some ofthese challenges, e.g., Maitra et al, Electrophoresis, 33: 3418-3428(2012); Venkatesan et al, Nature Nanotechnology, 6: 615-624 (2011);however, these approaches have their own set of technical difficulties,such as, reliable nanostructure fabrication, control of DNAtranslocation rates through nanopores, nucleotide discrimination,detection of electrical signals from large arrays of nanopore sensors,and the like, e.g. Branton et al, Nature Biotechnology, 26(10):1146-1153 (2008); Venkatcsan et al (cited above).

Optical detection of nucleotides has been proposed as a potentialsolution to some of the technical difficulties in the field of nanoporesequencing, e.g. Huber, International patent publication WO 2011/040996;Russell, U.S. Pat. No. 6,528,258; Pittaro, U.S. patent publication2005/0095599; Joyce, U.S. patent publication 2006/0019259; Chan, U.S.Pat. No. 6,355,420; McNally et al, Nano Lett., 10(6): 2237-2244 (2010);and the like, and has been implemented in the field of single-moleculesequencing using arrays of zero mode waveguides, e.g. Eid et al,Science, 323: 133-138 (2009). However, a limitation of optically-basednanopore and zero mode waveguide sequencing relates to the resolutionlimits of optical detection systems. Although current nanoscalefabrication techniques are capable of producing arrays of sub-10 nmpores and wells with comparable pore-to-pore or well-to-well spacing,the full potential of such arrays cannot be used to advantageouslyachieve higher throughput rates because of the resolution limit of theoptical detection systems.

In view of the above, it would be advantageous to nanopore sensortechnology in general and its particular applications, such as opticallybased nanopore sequencing and/or zero mode waveguide sequencing, ifmethods were available for ameliorating the limitations imposed bydetection resolution limits.

SUMMARY OF THE INVENTION

The present invention is directed to devices and methods for efficientoptical detection and analysis of polymers, such as polynucleotides,using high density arrays of nanostructures, such as nanopores ornanowells.

In some embodiments, the invention is directed to methods and apparatusfor detecting sequences of optical signals from parallel reactions on anarray, wherein apparatus of the invention comprise the followingelements: (a) an array of nanostructures each comprising a reaction siteand each capable of confining a reaction that generates a sequence ofoptical signals, the nanostructures of the array being arranged inclusters each comprising a number of nanostructures and each differentcluster of nanostructures being disposed within a different resolutionlimited area; and (b) an optical system operatively associated with thearray for detecting optical signals from the reactions. In someembodiments, the number of nanostructures in each cluster is eithergreater than one or a random variable with an average value greater thanzero. In some of the foregoing embodiments, polymers being analyzedcomprise polynucleotides and such polynucleotides are translocatedthrough the nanopores electrophoretically from a “cis” chamber to a“trans” chamber.

In other embodiments, the invention includes a method of sequencingpolynucleotides each having a plurality of optical labels attached to asequence of nucleotides, the method comprising the following steps: (a)translocating single stranded polynucleotides at a concentration andflux through a nanopore array, wherein substantially every nucleotide ofeach single stranded polynucleotides has an optical label attached, theoptical label capable of generating an optical signal indicative of thenucleotide to which it is attached, and wherein the nanopore arraycomprises clusters of nanopores, such that nanopores of differentclusters are within different resolution limited areas; (b) exposing theoptical labels of each nucleotide to excitation radiation upon exiting ananopore; (c) measuring in each resolution limited region on thenanopore array optical signals generated by optical labels exitingnanopores to identify the nucleotide to which the optical label isattached whenever such optical signals are from a single optical label;and (d) determining a nucleotide sequence of the polynucleotide from asequence of optical signals from single optical labels. In someembodiments of the above, the pluralities of nanopores within theresolution limited areas, concentration of the polynucleotides, and/orflux of the polynucleotides through said nanopore array are selected tomaximize the number of sequence-able nanopores in the array. In otherembodiments of the above method, the flux and/or movement of the singlestranded polynucleotides through the nanopore array is controlled tomaximize the number of sequence-able nanopores in the array bycontrolling an electrical potential across the nanopore array. That is,in some embodiments, during a sequencing operation an electricalpotential across the nanopore array is varied in order to maximizesequencing throughput, e.g. by maximizing the total number ofsequence-able nanopores in the array.

The present invention is exemplified in a number of implementations andapplications, some of which are summarized below and throughout thespecification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrate the limitations of optical detection of nanoporeevents in face of signal resolution limits.

FIGS. 1B-1E illustrate various nanopore array configurations related tosignal resolution limits.

FIGS. 1F-1G illustrate analyte arrival times and signal acquisitionrates for resolution limit areas or regions.

FIGS. 2A-2C illustrate an embodiment of a hybrid nanopore.

FIG. 2D illustrate an embodiment of the nanopore of the invention withpositioning of a member of a FRET pair using oligonucleotidehybridization.

FIG. 2E illustrates one embodiment of a hybrid nanopore where thesurface of the solid state membrane (201) coated with a hydrophobiclayer (202) to which a lipid layer is adhered (203). The lipids forms agigaohm seal with the inserted nanopore protein.

FIG. 3 illustrates a nanopore device and method for use with the presentinvention.

FIGS. 4A-4F illustrate clusters of labeled protein nanopores disposed inlipid bilayers across apertures in a solid phase membrane.

DETAILED DESCRIPTION OF THE INVENTION

While the invention is amenable to various modifications and alternativeforms, specifics thereof have been shown by way of example in thedrawings and will be described in detail. It should be understood,however, that the intention is not to limit the invention to theparticular embodiments described. On the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention. For example, particular nanoporetypes and numbers, particular labels, FRET pairs, detection schemes,fabrication approaches of the invention are shown for purposes ofillustration. It should be appreciated, however, that the disclosure isnot intended to be limiting in this respect, as other types ofnanopores, arrays of nanopores, and other fabrication technologies maybe utilized to implement various aspects of the systems discussedherein. Guidance for aspects of the invention is found in many availablereferences and treatises well known to those with ordinary skill in theart, including, for example, Cao, Nanostructures & Nanomaterials(Imperial College Press, 2004); Levinson, Principles of Lithography,Second Edition (SPIE Press, 2005); Doering and Nishi, Editors, Handbookof Semiconductor Manufacturing Technology, Second Edition (CRC Press,2007); Sawyer et al, Electrochemistry for Chemists, 2^(nd) edition(Wiley Interscience, 1995); Bard and Faulkner. Electrochemical Methods:Fundamentals and Applications, 2^(nd) edition (Wiley, 2000); Lakowicz,Principles of Fluorescence Spectroscopy, 3^(rd) edition (Springer,2006); Hermanson, Bioconjugate Techniques, Second Edition (AcademicPress, 2008); and the like, which relevant pans are hereby incorporatedby reference.

The invention is directed to methods and devices for efficient polymeranalysis using dense arrays of nanoscale structures, such as nanopores,nanowells, nanoparticles, or the like, coupled with optical detectionsystems. In some embodiments, polymers of interest are linear polymerscomprising sequences of at least two different kinds of monomer linkedin a linear chain with substantially every one of at least one kind ofmonomer being labeled with an optical label capable of generating anoptical signal indicative of the monomer to which it is attached. Inother embodiments, polymers of interest are linear polymers comprisingsequences of at least two different kinds of monomer linked in a linearchain with substantially every monomer of each kind being labeled withan optical label capable of generating an optical signal indicative ofthe monomer to which it is attached. In still other embodiments,polymers of interest are polynucleotides which capable of participatingin a reaction that generates a sequence of optical signals that containsinformation about the nucleotide sequence of such polynucleotide.Polymers of particular interest are polynucleotides, especially singlestranded DNAs. Also of particular interest are DNA polymers whosenucleotides are labeled with fluorescent dyes, such as fluorescent dyesfrom a mutually quenching set. In one aspect, the invention providesmethods and devices to determine monomer sequences of complete orpartial polymers in a sample. In another aspect, the invention providesmethods to determine an optical signature, or fingerprint, of a polymeror polynucleotide in a sample. Methods and devices of the inventionaddress the problem of efficient use of high capacity nanopore ornanowell arrays in view of the resolution limits of optical detection.Methods and devices of the invention also address the problem of loss ofdata acquisition capability caused by nonfunctional nanopores ornanowells, which may be caused by a variety of conditions, including butnot limited to, membrane failures or defects from fabrication errors,inactive enzymes, such as, inactive or mis-attached polymerases, andwhen protein nanopores are employed, protein mis-folding, subunitmis-aggregation, inoperable orientation of the protein nanopore in amembrane, and the like.

As used herein, a “resolution limited area” is an area of a surface of ananopore or nanowell array within which individual features or lightemission sources cannot be distinguished by an optical signal detectionsystem. Without intending to be limited by theory, such resolutionlimited area is determined by a resolution limit (also sometimesreferred to as a “diffraction limit” or “diffraction barrier”) of anoptical system. Such limit is determined by the wavelength of theemission source and the optical components and may be defined by d=λ/NA,where d is the smallest feature that can be resolved, λ is thewavelength of the light and NA is the numerical aperture of theobjective lens used to focus the light. Thus, whenever two or morenanopores are within a resolution limited area and two or more opticalsignals are generated at the respective nanopores, an optical detectionsystem cannot distinguish or determine which optical signals came fromwhich nanopore. In accordance with the invention, a surface of ananopore array may be partitioned, or subdivided, into non-overlappingregions, or substantially non-overlapping regions, corresponding toresolution limited areas. The size of such subdivisions corresponding toresolution limited areas may depend on a particular optical detectionsystem employed. In some embodiments, whenever light emission sourcesare within the visible spectrum, a resolution limited area is in therange of from 300 nm² to 3.0 μm²; in other embodiments, a resolutionlimited area is in the range of from 1200 nm, to 0.7 μm²; in otherembodiments, a resolution limited area is in the range of from 3×10⁴ nm²to 0.7 μm², wherein the foregoing ranges of areas are in reference to asurface of a nanopore or nanowell array. In some embodiments, thevisible spectrum means wavelengths in the range of from about 380 nm toabout 700 nm.

An average number of active nanopores in a nanopore array may beestimated as follows. Let a be the fraction of nanopores that arefunctional; let t be the average transit time of a polymer through ananopore; and let w be the average wait time to the next polymer, orpolymer fragment. If a nanopore is defined as “active” whenever it iscurrently translocating a polymer, then an expression for the activefraction of nanopores, u, may be given as follows:u=αt(w+t)This expression suggests that u could be increased by increasing α or tor by reducing, w. For example, α could be increased by improvingmanufacture of the nanopore arrays, t could be increased by increasingthe length of polymer analyzed, and w could be reduced by increasing theflux of polymer across the nanopore array, for example, by increasingconcentration of polymer and/or increasing the polymer driving force,e.g. an electric potential across the array, in the case of DNA. Asnoted above, these approaches to increasing the efficiency of a nanoporearray are limited because, although they may lead to a greater number ordensity of active nanopores, whenever two or more nanopores are activewithin a resolution limited area the resulting signals do not provideuseful information. Therefore, in one aspect, the method of theinvention provides values of u that maximize the number of nanoporesproviding useful information in view of constraints imposed by thelimits on resolution. In one aspect, nanopores capable of providinguseful sequence information are those which are the only active nanoporewithin a resolution limited area, referred to herein as “sequence-ablenanopore.” That is, a sequence-able nanopore is one which does not haveother active nanopores within the same resolution limited area at thesame time. For nanopores spaced in a square array, the numbersequence-able nanopores may be estimated as follows.

Assume an embodiment with a square of clusters of nanopores of area, A,with distance, d, between clusters, where d is the diffraction limitthat defines a resolution limited area. The number of clusters is thenA/d²=4270 clusters. The fraction, p1, of clusters that have exactly oneactive nanopore (i.e. exactly one sequence-able nanopore) is given byp1=ku(1−u)^(k-1), where again u is the active fraction of nanopores. Thetotal number of sequence-able pores is on average, Ap1/d², orkAu(1−u)^(k-1)/d². For example, if u is fixed at u=0.2, the foregoingexpression for p1 is maximized with k=4 and k=5. For illustration, if uis fixed at u=0.2 and the number of clusters is 1000, the value of p1 ismaximized when k=4 or k=5. Taking k=4, the total number of pore will be4000, and the number of sequence-able pores will be 410 on average. Forcomparison, a grid of single pores with the same spacing d will havejust 1000 pores, and the number of sequence-able pores will be only 200on average. Thus in the example the invention achieves more than doublethe throughput of an ordinary grid.

FIG. 1A, which is not intended to represent the actual physical orchemical condition adjacent to a nanopore array, shows two solid phasemembranes: membrane (100) having a single nanopore within resolutionlimited area (101) and membrane (102) having a plurality, K, ofnanopores within resolution limited area (103). Below, membranes (100)and (102) are shown polymers (108) consisting of two differently labeledmonomers (represented as linked black and white circles). Polymers (108)have an average length, concentration and flux across membranes (100)and (102). A flux of polymers (108) may be produced by a variety ofmethods, for example, when polymers are polynucleotides, a flux may beproduced by an electric field. Optical labels on monomers of polymers(108) are excited to generate optical signals as each monomer exits froma nanopore of membranes (100) and (102). For example, when opticallabels are fluorescent labels, such as FRET acceptors, such excitationmay be accomplished using a total internal reflectance fluorescence(TIRF) microscope system as a detection system, as illustrateddiagrammatically by (114). Fluorescent labels may be excited directly orindirectly using FRET donor-acceptor pairs. For each resolution limitedarea, detectors (115) and (116) collect optical signals and convert theminto values that can be displayed, such as the curves representingoptical signals from resolution limited area X (111) and the curvesrepresenting optical signals from resolution limited area Y (113). Insome embodiments, an objective of the analytical systems illustrated inFIG. 1A is to use the optical signals generated by the optical labels toidentify sequences of monomers among polymers (108). This may beaccomplished readily in the case where a single operable nanopore iswithin a resolution limited area, as shown by (101). However, when morethan one nanopore is within a resolution limited area and more than onepolymer translocates through such nanopores causing multiple opticalsignals to be generated, the recorded signals, such as (113), loseinformation from which monomer sequences can be determined. Thus, forexample, in the single nanopore case, base (or monomer) calls can bemade successfully in a 2-label system; that is, A, B (i.e. “not-A”), Band A (110); whereas, in the multiple nanopore case, base calls cannotbe made successfully; that is, the calls would be A, N, B and N, wherethe two “N” calls correspond to mixed optical signals whose componentscannot be assigned to a particular nanopore within resolution limitedarea (103).

As mentioned above, the problem of multiple nanopores within aresolution limited area may be addressed by fabricating nanopore arraysso that the density of nanopores is sufficiently low that there is amaximum of a single nanopore within each resolution limited area, asillustrated in FIG. 1B. There nanopores (131) of a portion of nanoporearray (130) are spaced (136) so that there is only one nanopore within aresolution limited area (134) having diameter (139). Unfortunately, suchan approach is not efficient and does not take advantage of fabricationcapabilities that permit much higher densities of nanopores, asillustrated in FIG. 1C, where nanopores (131) of a portion of nanoporearray (132) are spaced (137) so that there multiple nanopores within aresolution limited area (134). The lack of efficiency is particularlyexacerbated when hybrid nanopores are employed that comprise a solidphase membrane with fabricated apertures containing protein nanoporesimmobilized therein, e.g. as described in Huber et al, U.S. patentpublication US2013/0203050, which is incorporated herein by reference.Although hybrid nanopores are very useful because of the highly regularbores or lumens of the immobilized proteins, only a fraction of theimmobilized protein nanopores may be functional, or operable; that is,only a fraction have unobstructed bores that provide a fluidcommunication path between two chambers separated by a solid phasemembrane (referred to herein as an “operable fraction” or “functionalfraction”). Typically protein nanopores of an array of hybrid nanoporeshave an operable fraction in the range of from 10-50 percent, andfrequently have an operable fraction of about 25 percent. For suchnanopore arrays, the inefficiency of the arrangement shown in FIG. 1B isreadily apparent: a large fraction of the array would be inoperable andtherefore useless.

This problem may be addressed by providing arrays of clusters ofnanopores, as illustrated by FIG. 1D (showing a rectilinear array ofclusters) and 1E (showing a hexagonal array of clusters). In part, thepresent invention is based on the recognition and appreciation thatsequencing throughput per unit area of nanopore array may be increasedby employing nanopore arrays that are arrays of clusters of nanoporeswherein each cluster contains a plurality of nanopores and wherein thecluster-to-cluster distance is approximately equal to the diffractionlimit distance. Stated another way, nanopores in different clusters arein different resolution limited areas. One of ordinary skill wouldrecognize that this principle may be applied to any array ofnanostructures, such as nanowells or nanoparticles, that are used togenerate sequences of optical signals. Returning to FIGS. 1D and 1E,portion of nanopore array (140) shows clusters (141) of a plurality ofnanopores (or apertures) (131), where clusters (141) are spaced with aninter-cluster distance (142) sufficiently great that each cluster may bewithin a separate resolution limited area (144) and nanopores ofdifferent clusters do not share a resolution limited area. In oneembodiment, a plurality of nanopores is selected so that the averagenumber of operable nanopores is between 1 and 2. In another embodiment,the plurality of nanopores within a cluster is in the range of from 2 to9; in still another embodiment, the plurality of hybrid nanopores withina cluster is in the range of from 2 to 6. Likewise, clusters ofnanopores may be arranged into a hexagonal array (140) as shown in FIG.1E. In such an array, surface (195), for example, of a solid phasemembrane, is partitioned into hexagonal regions (for example, 190)having areas substantially equal to resolution limited areas (192) andeach containing a cluster of nanopores (194). In some embodiments, inarrays of clusters of nanostructures, inter-nanostructure distanceswithin a cluster may be in the range of from 10-200 nm and inter-clusterdistances (for example, a center-to-center distance) may be at least 500nm, or at least 1 μm. In other embodiments, inter-cluster distances maybe in the range of from 500 nm to 10 μm, or from 1 μm to 10 μm.

In some embodiments, clusters may also be formed by disposing proteinnanopores in lipid bilayers supported by solid phase membrane containingan array (4000) of apertures, for example, as illustrated in FIG. 4F.For example, array (4000) may comprise apertures fabricated (e.g.drilled, etched, or the like) in solid phase support (4100 in FIG. 4Fand 4102 in FIG. 4A). The geometry of such apertures may vary dependingon the fabrication techniques employed. For example, such apertures(4202) in FIG. 4F are depicted as circular and aperture (4202) in FIG.4E is depicted as rectangular. In some embodiments, each such apertureis associated with, or encompassed by, a separate resolution limitedarea (4244), as illustrated in FIG. 4F; however, in other embodiments,multiple apertures may be within the same resolution limited area. Thecross-sectional area of the apertures may vary widely and may or may notbe the same as between different clusters, although such areas areusually substantially the same as a result of conventional fabricationapproaches. In some embodiments, apertures have a minimal lineardimension (4103) (e.g. diameter in the case of circular apertures) inthe range of from 10 to 200 nm, or have areas in the range of from about100 to 3×10⁴ nm². Across the apertures is disposed a lipid bilayer,illustrated in cross-section in FIGS. 4A-4D. In some embodiments, suchlipid bilayer (4120) is disposed over one surface of solid phasemembrane (4100). In some embodiments, protein nanopores (4104 in FIGS.4A-4F) are inserted into portions of lipid bilayer (4120) spanning theapertures, where in some embodiments, such as those depicted, proteinnanopores may be directly labeled (4127), e.g. with a FRET donor. Insome embodiments, such protein nanopores are inserted from solution in achamber on one side of solid phase membrane (4100), which results in arandom placement of protein nanopores into the aperture, such thatdifferent apertures may receive different numbers of protein nanopores,as illustrated in FIGS. 4A-4D, where apertures are shown with no, one,two, or three protein nanopores. The distribution of protein nanoporesper aperture may be varied, for example, by controlling theconcentration of protein nanopores during inserting step. As illustratedin FIG. 4F, in such embodiments, clusters of nanopores may comprise arandom number of nanopores, for example, as shown by the representativeclusters (1 through 4) where cluster 1 contains a single proteinnanopore (4104), cluster 2 contains no protein nanopore, cluster 3contains two protein nanopores, and cluster 4 contains four proteinnanopores. In some embodiments, in which protein nanopores insertrandomly into apertures, clusters containing one or more apertures onaverage have a number of protein nanopores that is greater than zero; inother embodiments, such clusters have a number of protein nanopores thatis greater than 0.25; in other embodiments, such clusters have a numberof protein nanopores that is greater than 0.5; in other embodiments,such clusters have a number of protein nanopores that is greater than0.75; in other embodiments, such clusters have a number of proteinnanopores that is greater than 1.0.

The effect of using arrays of clusters may be illustrated by consideringthe expected number of operable nanopores per unit area, such as area(133) in FIG. 1B containing four nanopores, each in a separateresolution limited area. In this configuration, if the fraction ofoperable nanopores is 0.25, then the expected number of operablenanopores in area (133) is simply E=4×(0.25)=1. With clusters of 2nanopores the expected number of operable nanopores per cluster followsa binomial distribution as follows: E=0×P[n=0]+1×P[n=1]+2×P[n=2], whereP[n=i] is the probability that a cluster has i operable nanopores. Thus,for clusters of 2 nanopores, the expected number of operable nanoporesis 0.5, when the operability rate is 0.25.

FIG. 1F shows solid phase membrane (150) with an array of nanopores(152) spaced so that there is only a single nanopore within resolutionlimited area (154) (a configuration similar to that of (100) of FIG.1A). At any given nanopore entrance, polymers (153) will be captured(155) at random times. For example, the arrival of such polymers (155)may be modeled as Poisson counting process wherein the sequence ofinterarrival times is exponentially distributed, which roughly means thehigher the concentration and flux of polymers the shorter theinterarrival time. In FIG. 1G (illustrating signals (181 a, 181 b, 181c) generated by a sequence of captured polymers processed by ananopore), if it is assumed that each polymer has the same length, thenpolymer processing time (or equivalently nanopore occupancy time) may berepresented as constant length segments (182). Times between polymers(183) are the wait times during which a nanopore is inactive and notgenerating optical signals. After a polymer completes its translocationof a nanopore, the nanopore ceases to generate optical signals for atime until the next polymer arrives. The duty cycle of a single nanoporeper resolution limited area may approach 100 percent by raising theconcentration and/or flux, thereby forcing wait times to approach zero,but the information rate, because there is only one nanopore perresolution limited area, obtained per unit area of nanopore array islimited. In the same area a higher rate of information acquisition ispossible by using multiple nanopores per resolution limited area byadjusting polymer concentration, magnitude of polymer flux to the solidphase membrane, and average polymer length.

In some embodiments, the invention is directed to methods of determininga nucleotide sequence of at least one polynucleotide which comprises thefollowing steps: (a) translocating single stranded polynucleotides at aconcentration and flux through a nanopore array, wherein substantiallyevery nucleotide of each single stranded polynucleotides is labeled withan optical label capable of generating an optical signal indicative ofthe nucleotide to which it is attached, and wherein the nanopore arraycomprises clusters of nanopores, each cluster comprising a plurality ofnanopores within a resolution limited area such that nanopores ofdifferent clusters are within different resolution limited areas; (b)exposing the optical labels of each nucleotide to excitation radiationupon exiting a nanopore; (c) measuring in each resolution limited regionon the nanopore array optical signals generated by optical labelsexiting nanopores to identify the nucleotide to which the optical labelis attached whenever such optical signals are from a single opticallabel; and (d) determining a nucleotide sequence of the polynucleotidefrom a sequence of optical signals from single optical labels. Infurther embodiments, the density or magnitude of the pluralities ofnanopores within resolution limited areas, target polynucleotideconcentration, and/or flux of target polynucleotide through the nanoporearray are selected to maximize the number of sequence-able nanopores inthe array.

Nanopores and Nanopore Sequencing

Nanopores used with the invention may be solid-state nanopores, proteinnanopores, or hybrid nanopores comprising protein nanopores or organicnanotubes such as carbon nanotubes, configured in a solid-statemembrane, or like framework. Important features of nanopores include (i)constraining analytes, particularly polymer analytes, to pass through adetection zone in sequence, or in other words, so that monomers pass adetection zone one at a time, or in single file, (ii) compatibility witha translocating means (if one is used), that is, whatever method is usedto drive an analyte through a nanopore, such as an electric field, andoptionally, (iii) suppression of fluorescent signals within the lumen,or bore, of the nanopore. In some embodiments, nanopores used inconnection with the methods and devices of the invention are provided inthe form of arrays, such as an array of clusters of nanopores, which maybe disposed regularly on a planar surface. In some embodiments, clustersare each in a separate resolution limited area so that optical signalsfrom nanopores of different clusters are distinguishable by the opticaldetection system employed, but optical signals from nanopores within thesame cluster cannot necessarily be assigned to a specific nanoporewithin such cluster by the optical system employed.

Nanopores may be fabricated in a variety of materials including but notlimited to, silicon nitride (Si₃N₄), silicon dioxide (SiO₂), and thelike. The fabrication and operation of nanopores for analyticalapplications, such as DNA sequencing, are disclosed in the followingexemplary references that are incorporated by reference: Russell, U.S.Pat. No. 6,528,258; Feier. U.S. Pat. No. 4,161,690; Ling, U.S. Pat. No.7,678,562; Hu et al, U.S. Pat. No. 7,397,232; Golovchenko et al, U.S.Pat. No. 6,464,842; Chu et al, U.S. Pat. No. 5,798,042; Saner et al,U.S. Pat. No. 7,001,792; Su et al, U.S. Pat. No. 7,744,816; Church etal, U.S. Pat. No. 5,795,782; Bayley et al, U.S. Pat. No. 6,426,231;Akeson et al, U.S. Pat. No. 7,189,503; Bayley et al, U.S. Pat. No.6,916,665; Akeson et al, U.S. Pat. No. 6,267,872; Meller et al, U.S.patent publication 2009/0029477; Howorka et al, International patentpublication WO2009/007743; Brown et al, International patent publicationWO2011/067559; Meller et al, International patent publicationWO2009/020682; Polonsky et al, International patent publicationWO2008/92760; Van der Zaag et al, International patent publicationWO2010/007537; Yan et al, Nano Letters, 5(6): 1129-1134 (2005); Iqbal etal, Nature Nanotechnology, 2: 243-248 (2007); Wanunu et al, NanoLetters, 7(6): 1580-1585 (2007); Dekker, Nature Nanotechnology, 2:209-215 (2007); Storm et al, Nature Materials, 2: 537-540 (2003); Wu etal, Electrophoresis, 29(13): 2754-2759 (2008); Nakane et al,Electrophoresis, 23: 2592-2601 (2002); Zhe et al, J. Micromech.Microeng., 17: 304-313 (2007); Henriquez, et al, The Analyst, 129:478-482 (2004); Jagtiani et al, J. Micromech. Microeng., 16: 1530-1539(2006); Nakane et al, J. Phys. Condens. Matter, 15 R1365-R1393 (2003);DeBlois et al, Rev. Sci. Instruments, 41(7); 909-916 (1970); Clarke etal, Nature Nanotechnology, 4(4): 265-270 (2009); Bayley et al, U.S.patent publication 2003/0215881; and the like.

Briefly, in some embodiments, a 1-50 nm channel or aperture is formedthrough a substrate, usually a planar substrate, such as a membrane,through which an analyte, such as single stranded DNA, is induced totranslocate. The solid-state approach of generating nanopores offersrobustness and durability as well as the ability to tune the size andshape of the nanopore, the ability to fabricate high-density arrays ofnanopores on a wafer scale, superior mechanical, chemical and thermalcharacteristics compared with lipid-based systems, and the possibilityof integrating with electronic or optical readout techniques. Biologicalnanopores on the other hand provide reproducible narrow bores, orlumens, especially in the 1-10 nanometer range, as well as techniquesfor tailoring the physical and/or chemical properties of the nanoporeand for directly or indirectly attaching groups or elements, such asfluorescent labels, which may be FRET donors or acceptors, byconventional protein engineering methods. Protein nanopores typicallyrely on delicate lipid bilayers for mechanical support, and thefabrication of solid-state nanopores with precise dimensions remainschallenging. In some embodiments, solid-state nanopores may be combinedwith a biological nanopore to form a so-called “hybrid” nanopore thatovercomes some of these shortcomings, thereby providing the precision ofa biological pore protein with the stability of a solid state nanopore.For optical read out techniques a hybrid nanopore provides a preciselocation of the nanopore which simplifies the data acquisition greatly.

In some embodiments, arrays of clusters of nanopores of the inventionmay be used with a method for analyzing one or more polymer analytescomprising the following steps: (a) translocating a polymer analytethrough a nanopore having a bore and an exit, the polymer analytecomprising a sequence of monomers, wherein substantially each monomer islabeled with a fluorescent label such that fluorescent labels ofadjacent monomers are in a quenched state by self-quenching one anotheroutside of the nanopore and fluorescent labels are in a stericallyconstrained state and incapable of generating a detectable fluorescentsignal inside of the nanopore; (b) exciting each fluorescent label atthe exit of the nanopore as it transitions from a sterically constrainedstate to a quenched state so that a fluorescent signal is generatedwhich is indicative of the monomer to which it is attached; (c)detecting the fluorescent signal to identify the monomer. As usedherein, “substantially every”, “substantially all”, or like terms, inreference to labeling monomers, particularly nucleotides, acknowledgesthat chemical labeling procedures may not result in complete labeling ofevery monomer; to the extent practicable, the terms comprehend thatlabeling reactions in connection with the invention are continued tocompletion; in some embodiments, such completed labeling reactionsinclude labeling at least fifty percent of the monomers; in otherembodiments, such labeling reactions include labeling at least eightypercent of the monomers; in other embodiments, such labeling reactionsinclude labeling at least ninety-five percent of the monomers; in otherembodiments, such labeling reactions include labeling at leastninety-nine percent of the monomers.

In another embodiment, arrays of clusters of nanopores of the inventionmay be used with a method for analyzing one or more polymer analytescomprising the following steps: (a) attaching a fluorescent labelsubstantially every monomer of one or more polymer analytes such thatfluorescent labels of adjacent monomers are in a quenched state, (b)translocating the polymer analytes through nanopores so that monomers ofeach polymer analyte traverses the nanopore in single file and whereineach nanopore has a bore and an exit, the bore sterically constrainingthe fluorescent labels in a constrained state so that no fluorescentsignal is generated therefrom inside the bore; (c) exciting during atransition interval each fluorescent label at the exit of the nanoporeas each fluorescent label transitions from a sterically constrainedstate to a quenched state, thereby generating a fluorescent signal thatis indicative of the monomer to which it is attached; (c) detecting thefluorescent signal to identify the monomer.

In another embodiment, arrays of clusters of nanopores of the inventionmay be used with a device for analyzing one or more labeled polymeranalytes, such as a device for determining a nucleotide sequence of oneor more labeled polynucleotide analytes, such device comprising thefollowing elements: (a) a solid phase membrane separating a firstchamber and a second chamber, the solid phase membrane having at leastone nanopore fluidly connecting the first chamber and the second chamberthrough a bore or lumen, the bore or lumen having a cross-sectionaldimension such that labels of a labeled polymer translocatingtherethrough are sterically constrained so that detectable signals arenot generated, and so that the labels of adjacent monomers of thelabeled polymer are self-quenching; (b) an excitation source forexciting each label when it exits the nanopore and enters the secondchamber so that a signal is generated indicative of a monomer to whichthe label is attached; and (c) a detector for collecting at least aportion of the signal generated by each excited label; and (d)identifying the monomer to which the excited label is attached by thecollected signal.

In some embodiments, methods and devices of the invention comprise asolid phase membrane, such as a SiN membrane, having an array ofapertures therethrough providing communication between a first chamberand a second chamber (also sometimes referred to as a “cis chamber” anda “trans chamber”) and supporting a lipid bilayer on a surface facingthe second, or trans, chamber. In some embodiments, diameters of theaperture in such a solid phase membrane may be in the range of 10 to 200nm, or in the range of 20 to 100 nm. In some embodiments, such solidphase membranes further include protein nanopores inserted into thelipid bilayer in regions where such bilayer spans the apertures on thesurface facing the trans chamber. In some embodiments, such proteinnanopores are inserted from the cis side of the solid phase membraneusing techniques described herein. In some embodiments, such proteinnanopores have a structure identical to, or similar to, α-hemolysin inthat it comprises a barrel, or bore, along an axis and at one end has a“cap” structure and at the other end has a “stem” structure (using theterminology from Song et al, Science, 274: 1859-1866 (1996)). In someembodiments using such protein nanopores, insertion into the lipidbilayer results in the protein nanopore being oriented so that its capstructure is exposed to die cis chamber and its stem structure isexposed to the trans chamber.

In some embodiments, methods and devices of the invention comprisedroplet interface bilayers, either as single droplets or as arraysdroplets, for example, as disclosed in Bayley et al, U.S. patentpublication 2014/0356289; Huang et al, Nature Nanotechnology,10.1038/nnano.2015.189. [Epub ahead of print]; or like reference, whichare hereby incorporated by reference. Briefly, protein nanopores (1.2nM) are placed in a 200-350 nl droplet (for example, 1.32 M KCl, 8.8 mMHEPES, 0.4 mM EDTA, pH 7.0 (αHL) or 8.0 (MspA), and incubated in, forexample, 3 mM 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) inhexadecane to form a lipid monolayer coating. A droplet may then betransferred by pipetting onto a coverslip in a measurement chamber, forexample, that permits application of voltages to move analytes andoptical detection, for example, by TIRF. The coverslip may be spincoated (3,000 r.p.m., 30 s) with a thin layer (˜200 nm) of agarose (0.66M CaCl2, 8.8 mM HEPES, pH 7.0 (αHL)/8.0 (MspA)) and subsequentlyincubated with 3 mM DPhPC in hexadecane. On contact with the monolayeron the agarose, a lipid coated droplet spontaneously forms a dropletinterface bilayer. A ground electrode (Ag/AgCl) may be inserted into thedroplet, with a corresponding active electrode (Ag/AgCl) in thesubstrate agarose. Voltage protocols may be applied with a patch clampamplifier (for example, Axopatch 2008, Molecular Devices). Nanoporespresent in the droplet spontaneously insert into the droplet interfacebilayer, and the ion flux may be detected both electrically and/oroptically (for example, by way of an ion-sensitive dye, such as Fluo-8,or the like).

In some embodiments, the solid phase membrane may be treated with a lowenergy ion beam to bleach its autofluorescence, e.g. as described inHuber et al, U.S. patent publication 2013/0203050, which is incorporatedherein by reference.

FIGS. 2A-2C are diagrams of embodiments of hybrid biosensors. Ananometer sized hole (2102) is drilled into a solid-state substrate, orsolid phase membrane, (2103) which separates two chambers, orcompartments cis (2101) and trans (2107). A protein biosensor (e.g aprotein nanopore) (2104) attached to a charged polymer (2105), such as asingle stranded DNA, is embedded into the solid-state nanohole byelectrophoretic transport. In FIG. 1C the protein biosensor is inserted.In a nanometer sized hole which surface has a hydrophobic coating (2106)and a lipid layer (2109) attached thereto. A nanopore may have twosides, or orifices. One side is referred to as the “cis” side and facesthe (−) negative electrode or a negatively charged buffer/ioncompartment or solution. The other side is referred to as the “trans”side and faces the (+) electrode or a positively charged buffer/ioncompartment or solution. A biological polymer, such as a labeled nucleicacid molecule or polymer can be pulled or driven through the pore by anelectric field applied through the nanopore, e.g., entering on the cisside of the nanopore and exiting on the trans side of the nanopore. Inaccordance with the invention, such nanopores may be arranged in anarray of clusters of such nanopores.

FIG. 2D shows an embodiment of a hybrid nanopore wherein proteinnanopore (2104) inserted into an aperture drilled in a solid statemembrane (2103). Attached to the protein nanopore (2104) is anoligonucleotide (2108) to which a complementary secondaryoligonucleotide (2111) is hybridized. In some embodiments, secondaryoligonucleotide (2111) has one or more second members of a FRET pair(2110) attached to it. Alternatively, a member of a FRET pair may bedirectly attached to an amino acid of a protein nanopore, e.g. label(2123) of FIG. 2E. For example, a hemolysin subunit may be modified byconventional genetic engineering techniques to substitute a cysteine fora suitably located amino acid adjacent to the exit of the nanopore, e.g.the threonine 129. An oligonucleotide or members of a FRET pair may beattached via the thio group of the cysteine using conventional linkerchemistries, e.g. Hermanson (cited above).

In some embodiments, the present invention may employ hybrid nanoporesin clusters, particularly for optical-based nanopore sequencing ofpolynucleotides. Such nanopores comprise a solid-state orifice, oraperture, into which a protein biosensor, such as a protein nanopore, isstably inserted. A protein nanopore (e.g. alpha hemolysin) may beattached to a charged polymer (e.g. double stranded DNA) which in anapplied electric field may be used to guide a protein nanopore into anaperture in a solid-state membrane. In some embodiments, the aperture inthe solid-state substrate is selected to be slightly smaller than theprotein, thereby preventing it from translocating through the aperture.Instead, the protein will be embedded into the solid-state orifice. Thesolid-state substrate can be modified to generate active sites on thesurface that allow the covalent attachment of the plugged-in proteinbiosensor resulting in a stable hybrid biosensor.

The polymer attachment site in the protein nanopore can be generated byprotein engineering e.g. a mutant protein can be constructed that willallow the specific binding of the polymer. As an example, a cysteineresidue may be inserted at the desired position of the protein. Thecysteine can either replace a natural occurring amino acid or can beincorporated as an addition amino acid. Care must be taken not todisrupt the biological function of the protein. The terminal primaryamine group of a polymer (i.e. DNA) is then activated using ahetero-bifunctional crosslinker (e.g. SMCC). Subsequently, the activatedpolymer is covalently attached to the cysteine residue of the proteinbiosensor. In some embodiments, the attachment of the polymer to thebiosensor is reversible. By implementing a cleavable crosslinker, aneasily breakable chemical bond (e.g. an S—S bond) is introduced and thecharged polymer may be removed after insertion of the biosensor into thesolid-state aperture.

For someone skilled in the art it is obvious that a wide variety ofdifferent approaches for covalent or non-covalent attachment methods ofa charged polymer to the protein nanopore are possible and the abovedescribed approach merely serves as an example. The skilled artisan willalso realize that a variety of different polymers may be used as a dragforce, including, but not limited to, single or double stranded DNA,polyethyleneglycol (PEG), polyvinylpyrrolidone (PVP), poly-L-lysine,linear polysaccharides etc. It is also obvious that these polymers mayexhibit either a negative (−) or positive (+) charge at a given pH andthat the polarity of the electric field may be adjusted accordingly topull the polymer-biosensor complex into a solid-state aperture.

In some embodiments, a donor fluorophore is attached to the proteinnanopore. This complex is then inserted into a solid-state aperture ornanohole (for example, 3-10 nm in diameter) by applying an electricfield across the solid state nanohole, or aperture, until the proteinnanopore is transported into the solid-state nanohole to form a hybridnanopore. The formation of the hybrid nanopore can be verified by (a)the inserted protein nanopore causing a drop in current based on apartial blockage of the solid-state nanohole and by (b) the opticaldetection of the donor fluorophore.

Once stable hybrid nanopores have formed single stranded, fluorescentlylabeled (or acceptor labeled) DNA is added to the cis chamber (thechamber with the (+) electrode). The applied electric field forces thenegatively charged ssDNA to translocate through the hybrid nanoporeduring which the labeled nucleotides get in close vicinity of the donorfluorophore.

Solid state, or synthetic, nanopores may be prepared in a variety ofways, as exemplified in the references cited above. In some embodimentsa helium ion microscope may be used to drill the synthetic nanopores ina variety of materials, e.g. as disclosed by Yang et al, Nanotechnology,22: 285310 (2011), which is incorporated herein by reference. A chipthat supports one or more regions of a thin-film material, e.g. siliconnitride, that has been processed to be a free-standing membrane isintroduced to the helium ion microscope (HIM) chamber. HIM motorcontrols are used to bring a free-standing membrane into the path of theion beam while the microscope is set for low magnification. Beamparameters including focus and stigmation are adjusted at a regionadjacent to the free-standing membrane, but on the solid substrate. Oncethe parameters have been properly fixed, the chip position is moved suchthat the free-standing membrane region is centered on the ion beam scanregion and the beam is blanked. The HIM field of view is set to adimension (in μm) that is sufficient to contain the entire anticipatednanopore pattern and sufficient to be useful in future optical readout(i.e. dependent on optical magnification, camera resolution, etc.). Theion beam is then rastered once through the entire field of view at apixel dwell time that results in a total ion dose sufficient to removeall or most of the membrane autofluorescence. The field of view is thenset to the proper value (smaller than that used above) to performlithographically-defined milling of either a single nanopore or an arrayof nanopores. The pixel dwell time of the pattern is set to result innanopores of one or more predetermined diameters, determined through theuse of a calibration sample prior to sample processing. This entireprocess is repeated for each desired region on a single chip and/or foreach chip introduced into the HIM chamber.

In some embodiments, the solid-state substrate may be modified togenerate active sites on the surface that allow the covalent attachmentof the plugged in protein biosensor or to modify the surface propertiesin a way to make it more suitable for a given application. Suchmodifications may be of covalent or non-covalent nature. A covalentsurface modification includes a silanization step where an organosilanecompound binds to silanol groups on the solid surface. For instance, thealkoxy groups of an alkoxysilane are hydrolyzed to formsilanol-containing species. Reaction of these silanes involves foursteps. Initially, hydrolysis of the labile groups occurs. Condensationto oligomers follows. The oligomers then hydrogen bond with hydroxylgroups of the substrate. Finally, during drying or curing, a covalentlinkage is formed with the substrate with concomitant loss of water. Forcovalent attachment organosilanes with active side groups may beemployed. Such side groups consist of, but are not limited to epoxy sidechain, aldehydes, isocyanates, isothiocyanates, azides or alkynes (clickchemistry) to name a few. For someone skilled in the an it is obviousthat multiple ways of covalently attaching a protein to a surface arepossible. For instance, certain side groups on an organosilane may needto be activated before being capable of binding a protein (e.g. primaryamines or carboxyl side groups activated with anN-hydroxysuccinimidester).

Another way of attaching a protein to the solid surface may be achievedthrough affinity binding by having one affinity partner attached to theprotein and the second affinity partner being located on the solidsurface. Such affinity pairs consist of the group of, but are notlimited to biotin-strepavidin, antigen-antibody and aptamers and thecorresponding target molecules. In a preferred embodiment the surfacemodification of the solid state nanopore includes treatment with anorganosilane that renders the surface hydrophobic. Such organosilanesinclude but are not limited to, alkanesilanes (e.g.octadecyldimethylchlorosilane) or modified alkanesilanes such asfluorinated alkanesilanes with an alkane chain length of 5 to 30carbons. The hydrophobic surface may then treated with a dilute solutionof a lipid in pentane. After drying of the solvent and immersing thesurface in an aqueous solution the lipid will spontaneously form a layeron the surface.

In some embodiments, a layer of lipid on the solid surface may bebeneficial for the formation of a hybrid nanopore. The lipid layer onthe solid phase may reduce the leak current between protein and solidstate nanopore and it may increase the stability of the inserted proteinpore. Combining a low capacitance solid substrate as well as a lipidcoating of said substrate may render the hybrid nanopore system amenableto an electrical readout based on current fluctuations generated bytranslocation of DNA through the hybrid nanopore. To achieve electricalread out with such a system a means of decreasing the translocationspeed of unmodified DNA must be combined with a lipid coated hybridnanopore. Molecular motors such as polymerases or helicases may becombined with a hybrid nanopore and effectively reduce the translocationspeed of DNA through the hybrid nanopore. The lipids used for coatingthe surface are from the group of sphingolipids, phospholipids orsterols. A method and/or system for sequencing a biological polymer ormolecule (e.g., a nucleic acid) may include exciting one or more donorlabels attached to a pore or nanopore. A biological polymer may betranslocated through the pore or nanopore, where a monomer of thebiological polymer is labeled with one or more acceptor labels. Energymay be transferred from die excited donor label to the acceptor label ofthe monomer as, after the labeled monomer passes through, exits orenters the pore or nanopore. Energy emitted by the acceptor label as aresult of the energy transfer may be detected, where the energy emittedby the acceptor label may correspond to or be associated with a singleor particular monomer (e.g., a nucleotide) of a biological polymer. Thesequence of the biological polymer may then be deduced or sequencedbased on the detection of the emitted energy from the monomer acceptorlabel which allows for the identification of the labeled monomer. Apore, nanopore, channel or passage, e.g., an ion permeable pore,nanopore, channel or passage may be utilized in the systems and methodsdescribed herein.

In some embodiments, a nanopore may have one or more labels attached. Insome embodiments, the label is a member of a Forster Resonance EnergyTransfer (FRET) pair. Such labels may comprise organic fluorophores,chemiluminescent labels, quantum dots, metallic nanoparticles and/orfluorescent proteins. The nucleic acid may have one distinct label pernucleotide. The labels attached to the nucleotides may be selected fromthe group consisting of organic fluorophores, chemiluminescent labels,quantum dots, metallic nanoparticles and fluorescent proteins. The labelattachment site in the pore protein can be generated by proteinengineering e.g. a mutant protein can be constructed that will allow thespecific binding of the label. As an example, a cysteine residue may beinserted at the desired position of the protein which inserts a thiol(SH) group that can be used to attach a label. The cysteine can eitherreplace a natural occurring amino acid or can be incorporated as anaddition amino acid. A malemeide-activated label is then covalentlyattached to the thiol residue of the protein nanopore. In a preferredembodiment the attachment of the label to the protein nanopore or thelabel on the nucleic acid is reversible. By implementing a cleavablecrosslinker, an easily breakable chemical bond (e.g. an S—S bond or a pHlabile bond) is introduced and the label may be removed when thecorresponding conditions are met.

A nanopore, or pore, may be labeled with one or more donor labels. Forexample, the cis side or surface and/or trans side or surface of thenanopore may be labeled with one or more donor labels. The label may beattached to the base of a pore or nanopore or to another portion ormonomer making up the nanopore or pore A label may be attached to aportion of the membrane or substrate through which a nanopore spans orto a linker or other molecule attached to the membrane, substrate ornanopore. The nanopore or pore label may be positioned or attached onthe nanopore, substrate or membrane such that the pore label can comeinto proximity with an acceptor label of a biological polymer, e.g., anucleic acid, which is translocated through the pore. The donor labelsmay have the same or different emission or absorption spectra. Thelabeling of a pore structure may be achieved via covalent ornon-covalent interactions.

A donor label (also sometimes referred to herein as a “pore label”) maybe placed as close as possible to the aperture (for example, at theexit) of a nanopore without causing an occlusion that impairstranslocation of a nucleic acid through the nanopore. A pore label mayhave a variety of suitable properties and/or characteristics. Forexample, a pore label may have energy absorption properties meetingparticular requirements. A pore label may have a large radiation energyabsorption cross-section, ranging, for example, from about 0 to 1000 nmor from about 200 to 500 nm. A pore label may absorb radiation within aspecific energy range that is higher than the energy absorption of thenucleic acid label, such as an acceptor label. The absorption energy ofthe pore label may be tuned with respect to the absorption energy of anucleic acid label in order to control the distance at which energytransfer may occur between the two labels. A pore label may be stableand functional for at least 10⁶ to 10⁹ excitation and energy transfercycles.

In some embodiments, a device for analyzing polymers each having opticallabels attached to a sequence of monomers may comprise the followingelements: (a) a nanopore array in a solid phase membrane separating afirst chamber and a second chamber, wherein nanopores of the nanoporearray each provide fluid communication between the first chamber and thesecond chamber and are arranged in clusters such that each differentcluster of nanopores is disposed within a different resolution limitedarea and such that each cluster comprises a number of nanopores that iseither greater than one or is a random variable with an average valuegreater than zero; (b) a polymer translocating system for movingpolymers in the first chamber to the second chamber through thenanopores of the nanopore array; and (c) a detection system forcollecting optical signals generated by optical labels attached topolymers whenever an optical label exits a nanopore within a resolutionlimited area.

Labels for Nanopores and Analytes

In some embodiments, a nanopore may be labeled with one or more quantumdots. In particular, in some embodiments, one or more quantum dots maybe attached to a nanopore, or attached to a solid phase support adjacentto (and within a FRET distance of an entrance or exit of a nanopore),and employed as donors in FRET reactions with acceptors on analytes.Such uses of quantum dots are well known and are described widely in thescientific and patent literature, such as, in U.S. Pat. Nos. 6,252,303;6,855,551; 7,235,361; and the like, which are incorporated herein byreference.

One example of a Quantum dot which may be utilized as a pore label is aCdTe quantum dot which can be synthesized in an aqueous solution. A CdTequantum dot may be functionalized with a nucleophilic group such asprimary amines, thiols or functional groups such as carboxylic acids. ACdTe quantum dot may include a mercaptopropionic acid capping ligand,which has a carboxylic acid functional group that may be utilized tocovalently link a quantum dot to a primary amine on the exterior of aprotein pore. The cross-linking reaction may be accomplished usingstandard cross-linking reagents (homo-bifunctional as well ashetero-bifunctional) which are known to those having ordinary skill inthe art of bioconjugation. Care may be taken to ensure that themodifications do not impair or substantially impair the translocation ofa nucleic acid through the nanopore. This may be achieved by varying thelength of the employed crosslinker molecule used to attach the donorlabel to the nanopore.

For example, the primary amine of the lysine residue 131 of the naturalalpha hemolysin protein (Song, L. et al., Science 274, (1996):1859-1866) may be used to covalently bind carboxy modified CdTe Quantumdots via 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride/N-hydroxysulfosuccinimide (EDC/NHS) coupling chemistry.Alternatively, amino acid 129 (threonine) may be exchanged intocysteine. Since there is no other cysteine residue in the natural alphahemolysin protein the thiol side group of the newly inserted cysteinemay be used to covalently attach other chemical moieties.

A variety of methods, mechanisms and/or routes for attaching one or morepore labels to a pore protein may be utilized. A pore protein may begenetically engineered in a manner that introduces amino acids withknown properties or various functional groups to the natural proteinsequence. Such a modification of a naturally occurring protein sequencemay be advantageous for the bioconjugation of Quantum dots to the poreprotein. For example, the introduction of a cysteine residue wouldintroduce a thiol group that would allow for the direct binding of aQuantum dot, such as a CdTe quantum dot, to a pore protein. Also, theintroduction of a Lysin residue would introduce a primary amine forbinding a Quantum dot. The introduction of glutamic acid or asparticacid would introduce a carboxylic acid moiety for binding a Quantum dot.These groups are amenable for bioconjugation with a Quantum dot usingeither homo- or hetero-bifunctional crosslinker molecules. Suchmodifications to pore proteins aimed at the introduction of functionalgroups for bioconjugation are known to those having ordinary skill inthe art. Care should be taken to ensure that the modifications do notimpair or substantially impair the translocation of a nucleic acidthrough the nanopore.

The nanopore label can be attached to a protein nanopore before or afterinsertion of said nanopore into a lipid bilayer. Where a label isattached before insertion into a lipid bilayer, care may be taken tolabel the base of the nanopore and avoid random labeling of the poreprotein. This can be achieved by genetic engineering of the pore proteinto allow site specific attachment of the pore label, as discussed below.An advantage of this approach is the bulk production of labelednanopores. Alternatively, a labeling reaction of a pre-inserted nanoporemay ensure site-specific attachment of the label to the base(trans-side) of the nanopore without genetically engineering the poreprotein.

A biological polymer, e.g., a nucleic acid molecule or polymer, may belabeled with one or more acceptor labels. For a nucleic acid molecule,each of the four nucleotides or building blocks of a nucleic acidmolecule may be labeled with an acceptor label thereby creating alabeled (e.g., fluorescent) counterpart to each naturally occurringnucleotide. The acceptor label may be in the form of an energy acceptingmolecule which can be attached to one or more nucleotides on a portionor on the entire strand of a converted nucleic acid.

A variety of methods may be utilized to label the monomers ornucleotides of a nucleic acid molecule or polymer. A labeled nucleotidemay be incorporated into a nucleic acid during synthesis of a newnucleic acid using the original sample as a template (“labeling bysynthesis”). For example, the labeling of nucleic acid may be achievedvia PCR, whole genome amplification, rolling circle amplification,primer extension or the like or via various combinations and extensionsof the above methods known to persons having ordinary skill in the art.

Labeling of a nucleic acid may be achieved by replicating the nucleicacid in the presence of a modified nucleotide analog having a label,which leads to the incorporation of that label into the newly generatednucleic acid. The labeling process can also be achieved by incorporatinga nucleotide analog with a functional group that can be used tocovalently attach an energy accepting moiety in a secondary labelingstep. Such replication can be accomplished by whole genome amplification(Zhang. L. et al., Proc. Natl. Acad. Sci. USA 89 (1992): 5847) or stranddisplacement amplification such as rolling circle amplification, nicktranslation, transcription, reverse transcription, primer extension andpolymerase chain reaction (PCR), degenerate oligonucleotide primer PCR(DOP-PCR) (Telenius, H. et al., Genomics 13 (1992): 718-725) orcombinations of the above methods.

A label may comprise a reactive group such as a nucleophile (amines,thiols etc.). Such nucleophiles, which are not present in naturalnucleic acids, can then be used to attach fluorescent labels via amineor thiol reactive chemistry such as NHS esters, maleimides, epoxy rings,isocyanates etc. Such nucleophile reactive fluorescent dyes (i.e.NHS-dyes) are readily commercially available from different sources. Anadvantage of labeling a nucleic acid with small nucleophiles lies in thehigh efficiency of incorporation of such labeled nucleotides when a“labeling by synthesis” approach is used. Bulky fluorescently labelednucleic acid building blocks may be poorly incorporated by polymerasesdue to steric hindrance of the labels during the polymerization processinto newly synthesized DNA.

In some embodiments. DNA can be directly chemically modified withoutpolymerase mediated incorporation of labeled nucleotides. One example ofa modification includes cis-platinum containing dyes that modify Guaninebases at their N7 position (Hoevel, T. et al., Bio Techniques 27 (1999):1064-1067). Another example includes the modifying of pyrimidines withhydroxylamine at the C6 position which leads to 6-hydroxylaminoderivatives. The resulting amine groups can be further modified withamine reactive dyes (e.g. NHS-Cy5). Yet another example are azide oralkyne modified nucleotides which are readily incorporated bypolymerases (Gierlich et al., Chem. Eur. J., 2007, 13, 9486-0404). Thealkyne or azide modified polynucleotide is subsequently labeled with anazide or alkyne modified fluorophore following well established clickchemistry protocols.

As mentioned above, in some embodiments, DNA may be labeled using “clickchemistry,” e.g. using commercially available kits (such as “Click-t”from Life Technologies, Carlsbad, Calif.). Click chemistry in generalrefers to a synthetic process in which two molecules are linked togetherby a highly efficient chemical reaction, one which is essentiallyirreversible, in which the yield is nearly 100%, and which produces fewor no reaction byproducts. More recently, the meaning has come to referto the cyclization reaction of a substituted alkyne with a substitutedazide to form a 1,2,3-triazole bearing the two substituents. Whencatalyzed by copper at room temperature the reaction is known as theHuisgen cycloaddition, and it fully satisfies the requirements for clickchemistry in that no other chemical functionality on the two moleculesis affected during the reaction. Thus the coupling reaction has foundbroad application in bioconjugate chemistry, for example, in dyelabeling of DNA or proteins, where many amine, hydroxy, or thiol groupsmay be found. The key requirement is that an alkyne group and an azidecan easily be introduced into the molecules to be coupled. For example,in the coupling of a fluorescent dye to a DNA oligonucleotide, the azidegroup is typically introduced synthetically into the dye, while thealkyne group is incorporated into the DNA during oligonucleotidesynthesis. Upon mixing in the presence of Cu+ the two components arequickly coupled to form the triazole, in this case bearing theoligonucleotide as one substituent and the dye as the other. Anothermore recent advance provides the alkyne component within a strained ringstructure. In this case the reaction with an azide does not require thecopper catalyst, being driven by release of the ring strain energy asthe triazole is formed. This is better known as the copper-free clickreaction. Guidance for applying click chemistry to methods of theinvention may be found in the following references which areincorporated by reference: Rostovtsev V V, Green L G; Fokin, Valery V,Sharpless K B (2002). “A Stepwise Huisgen Cycloaddition Process:Copper(I)-Catalyzed Regioselective “Ligation” of Azides and TerminalAlkynes”. Angewandie Chemie International Edition 41 (14): 2596-2599.Moses J E and Moorhouse A D (2007). “The growing applications of clickchemistry”. Chem. Soc. Rev. 36 (8): 1249-1262.

Whenever two or more mutually quenching dyes are used, such dyes may beattached to DNA using orthogonal attachment chemistries. For example,NHS esters can be used to react very specifically with primary amines ormaleimides will react with thiol groups. Either primary amines (NH₂) orthiol (SH) modified nucleotides are commercially available. Theserelatively small modifications are readily incorporated in a polymerasemediated DNA synthesis and can be used for subsequent labeling reactionsusing either NHS or maleimide modified dyes. Guidance for selecting andusing such orthogonal linker chemistries may be found in Hermanson(cited above).

Additional orthogonal attachment chemistries for typical attachmentpositions include Huisgen-type cycloaddition for a copper-catalyzedreaction and an uncatalyzed reaction; alkene plus nitrile oxidecycloaddition, e.g. as disclosed in Gutsmiedl et al, Org. Lett., 11:2405-2408 (2009); Diels-Alder cycloaddition, e.g. disclosed in Seelig etal, Tetrahedron Let., 38: 7729-7732 (1997); carbonyl ligation, e.g. asdisclosed in Casi et al, J. Am. Chem. Soc., 134: 5887-5892 (2012); Shaoet al J. Am. Chem. Soc., 17: 3893-3899 (1995); Rideout. Science, 233:561-563 (1986); Michael addition, e.g. disclosed in Brinkley.Bioconjugate Chemistry, 3: 2-13 (1992); native chemical ligation, e.g.disclosed in Schuler et al, Bioconjugate Chemistry, 13: 1039-1043(2002); Dawson et al, Science, 266: 776-779 (1994); or amide formationvia an active ester, e.g. disclosed in Hermanson (cited above).

A nucleic acid molecule may be directly modified with N-Bromosuccinimidewhich upon reacting with the nucleic acid will result in 5-Bromocystein,8-Bromoadenine and 8-Bromoguanine. The modified nucleotides can befurther reacted with di-amine nucleophiles. The remaining nucleophilecan then be reacted with an amine reactive dye (e.g. NHS-dye) (HermansonG. in Bioconjugate Techniques, cited above).

A combination of 1, 2, 3 or 4 nucleotides in a nucleic acid strand maybe exchanged with their labeled counterpart. The various combinations oflabeled nucleotides can be sequenced in parallel, e.g., labeling asource nucleic acid or DNA with combinations of 2 labeled nucleotides inaddition to the four single labeled samples, which will result in atotal of 10 differently labeled sample nucleic acid molecules or DNAs(G, A, T, C, GA, GT, GC, AT, AC, TC). The resulting sequence pattern mayallow for a more accurate sequence alignment due to overlappingnucleotide positions in the redundant sequence read-out. In someembodiments, a polymer, such as a polynucleotide or polypeptide, may belabeled with a single fluorescent label attached to a single kind ofmonomer, for example, every T (or substantially every T) of apolynucleotide is labeled with a fluorescent label. e.g. a cyanine dye.In such embodiments, a collection, or sequence, of fluorescent signalsfrom the polymer may form a signature or fingerprint for the particularpolymer. In some such embodiments, such fingerprints may or may notprovide enough information for a sequence of monomers to be determined.

In some embodiments, a feature of the invention is the labeling ofsubstantially all monomers of a polymer analyte with fluorescent dyes orlabels that are members of a mutually quenching set. The use of the term“substantially all” in reference to labeling polymer analytes is toacknowledge that chemical and enzymatic labeling techniques aretypically less than 100 percent efficient. In some embodiments.“substantially all” means at least 80 percent of all monomer havefluorescent labels attached. In other embodiments, “substantially all”means at least 90 percent of all monomer have fluorescent labelsattached. In other embodiments, “substantially all” means at least 95percent of all monomer have fluorescent labels attached.

A method for sequencing a polymer, such as a nucleic acid moleculeincludes providing a nanopore or pore protein (or a synthetic pore)inserted in a membrane or membrane like structure or other substrate.The base or other portion of the pore may be modified with one or morepore labels. The base may refer to the Trans side of the pore.Optionally, the Cis and/or Trans side of the pore may be modified withone or more pore labels. Nucleic acid polymers to be analyzed orsequenced may be used as a template for producing a labeled version ofthe nucleic acid polymer, in which one of the four nucleotides or up toall four nucleotides in the resulting polymer is/are replaced with thenucleotide's labeled analogue(s). An electric field is applied to thenanopore which forces the labeled nucleic acid polymer through thenanopore, while an external monochromatic or other light source may beused to illuminate the nanopore, thereby exciting the pore label. As,after or before labeled nucleotides of the nucleic acid pass through,exit or enter the nanopore, energy is transferred from the pore label toa nucleotide label, which results in emission of lower energy radiation.The nucleotide label radiation is then detected by a confocal microscopesetup or other optical detection system or light microscopy systemcapable of single molecule detection known to people having ordinaryskill in the art. Examples of such detection systems include but are notlimited to confocal microscopy, epifluorescent microscopy and totalinternal reflection fluorescent (TIRF) microscopy. Other polymers (e.g.,proteins and polymers other than nucleic acids) having labeled monomersmay also be sequenced according to the methods described herein. In someembodiments, fluorescent labels or donor molecules are excited in a TIRFsystem with an evanescent wave, sometimes referred to herein as“evanescent wave excitation.”

Energy may be transferred from a pore or nanopore donor label (e.g., aQuantum Dot) to an acceptor label on a polymer (e.g., a nucleic acid)when an acceptor label of an acceptor labeled monomer (e.g., nucleotide)of the polymer interacts with the donor label as, after or before thelabeled monomer exits, enters or passes through a nanopore. For example,the donor label may be positioned on or attached to the nanopore on thecis or trans side or surface of the nanopore such that the interactionor energy transfer between the donor label and acceptor label does nottake place until the labeled monomer exits the nanopore and comes intothe vicinity or proximity of the donor label outside of the nanoporechannel or opening. As a result, interaction between the labels, energytransfer from the donor label to the acceptor label, emission of energyfrom the acceptor label and/or measurement or detection of an emissionof energy from the acceptor label may take place outside of the passage,channel or opening running through the nanopore, e.g., within a cis ortrans chamber on the cis or trans sides of a nanopore. The measurementor detection of the energy emitted from the acceptor label of a monomermay be utilized to identify the monomer.

The nanopore label may be positioned outside of the passage, channel oropening of the nanopore such that the label may be visible or exposed tofacilitate excitation or illumination of the label. The interaction andenergy transfer between a donor label and acceptor label and theemission of energy from the acceptor label as a result of the energytransfer may take place outside of the passage, channel or opening ofthe nanopore. This may facilitate ease and accuracy of the detection ormeasurement of energy or light emission from the acceptor label, e.g.,via an optical detection or measurement device.

A donor label may be attached in various manners and/or at various siteson a nanopore. For example, a donor label may be directly or indirectlyattached or connected to a portion or unit of the nanopore.Alternatively, a donor label may be positioned adjacent to a nanopore.

Each acceptor labeled monomer (e.g., nucleotide) of a polymer (e.g.,nucleic acid) can interact sequentially with a donor label positioned onor next to or attached directly or indirectly to the exit of a nanoporeor channel through which the polymer is translocated. The interactionbetween the donor and acceptor labels may take place outside of thenanopore channel or opening, e.g., after the acceptor labeled monomerexits the nanopore or before the monomer enters the nanopore. Theinteraction may take place within or partially within the nanoporechannel or opening, e.g., while the acceptor labeled monomer passesthrough, enters or exits the nanopore.

When one of the four nucleotides of a nucleic acid is labeled, the timedependent signal arising from the single nucleotide label emission isconverted into a sequence corresponding to the positions of the labelednucleotide in the nucleic acid sequence. The process is then repeatedfor each of the four nucleotides in separate samples and the fourpartial sequences are then aligned to assemble an entire nucleic acidsequence.

When multi-color labeled nucleic acid (DNA) sequences are analyzed, theenergy transfer from one or more donor labels to each of the fourdistinct acceptor labels that may exist on a nucleic acid molecule mayresult in light emission at four distinct wavelengths or colors (eachassociated with one of the four nucleotides) which allows for a directsequence read-out.

Translocation Speed

A major obstacle associated with nanopore based sequencing approaches isthe high translocation velocity of nucleic acid through a nanopore(˜500,000-1,000,000 nucleotides/sec) which doesn't allow for directsequence readout due to the limited bandwidth of the recordingequipment. A way of slowing down the nucleic acid translocation with twodifferent nanopore proteins was recently shown by Cherf et al, (NatBiotechnol, 2012 Feb. 14; 30(4):344-8) and Manrao et al, (NatBiotechnol, 2012 Mar. 25; 30(4):349-53) and are incorporated herein byreference. Both groups used a DNA polymerase to synthesize acomplementary strand from a target template which resulted in thestep-wise translocation of the template DNA through the nanopore. Hence,the synthesis speed of the nucleic acid polymerase (10-500nucleotides/sec) determined the translocation speed of the DNA and sinceit's roughly 3-4 orders of magnitude slower than direct nucleic acidtranslocation the analysis of single nucleotides became feasible.However, the polymerase-aided translocation requires significant samplepreparation to generate a binding site for the polymerase and thenucleic acid synthesis has to be blocked in bulk and can only start oncethe nucleic acid-polymerase complex is captured by the nanopore protein.This results in a rather complex set-up which might prevent theimplementation in a commercial setting. Furthermore, fluctuation inpolymerase synthesis reactions such as a stalled polymerization as wellas the dissociation of the polymerase from the nucleic acid may hamperthe sequence read-out resulting in a high error rate and reducedread-length, respectively. In some embodiments, a target nucleic acid isenzymatically copied by incorporating fluorescent modified nucleotides.The resulting labeled nucleic acid has an increased nominal diameterwhich results in a decreased translocation velocity when pulled througha nanopore. The preferred translocation rate for optical sequencing liesin the range of 1-1000 nucleotides per second with a more preferredrange of 200-800 nucleotides per second and a most preferredtranslocation rate of 200-600 nucleotides per second.

Alternatively, translocation speed of a polynucleotide, especially asingle stranded polynucleotide, may be controlled by employing ananopore dimensioned so that adducts and/or labels, e.g. organic dyesattached to bases, inhibit but do not prevent polynucleotidetranslocation. A translocation speed may be selected by attaching labelsand/or adducts at a predetermined density. Such labels and/or adductsmay have regular spaced attachments, e.g. every third nucleotide or thelike, or they may have random, or pseudorandom attachments, e.g. every Cmay be labeled. In some embodiments, a selected number of differentnucleotides may be labeled, e.g. every A and C, or every A and G, orevery A and T, or every C, or the like, that results in an averagetranslocation speed. Such average speed may be decreased by attachingadducts to unlabeled nucleotides. Adducts include any molecule, usuallyand organic molecule, that may be attached to a nucleotide usingconventional chemistries. Typically adducts have a molecular weight inthe same range as common organic dyes, e.g. fluorescein. Cy3, or thelike. Adducts may or may not be capable of generating signals, that is,serving as a label. In some embodiments, adducts and/or labels areattached to bases of nucleotides. In other embodiments, labels and/oradducts may be attached to linkages between nucleosides in apolynucleotide. In one aspect, a method of controlling translocationvelocity of a single stranded polynucleotide through a nanoporecomprises the step of attaching adducts to the polynucleotide at adensity, wherein translocation velocity of the single strandedpolynucleotide monotonically decreases with a larger number of adductsattached, or with the density of adducts attached. In some embodiments,not every kind of nucleotide of a polynucleotide is labeled. Forexample, four different sets of a polynucleotide may be produced wherenucleotides of each set are labeled with the same molecule, e.g. afluorescent organic dye acceptor, but in each set a different kind ofnucleotide will be labeled. Thus, in set 1 only A's may be labeled; inset 2 only C's may be labeled: in set 3 only G's may be labeled: and soon. After such labeling, the four sets of polynucleotides may then beanalyzed separately in accordance with the invention and a nucleotidesequence of the polynucleotide determined from the data generated in thefour analysis. In such embodiments, and similar embodiments, e.g. twolabels are used, where some of the nucleotides of a polynucleotide arenot labeled, translocation speed through a nanopore will be affected bythe distribution of label along the polynucleotide. To prevent suchvariability in translocation speed, in some embodiments, nucleotidesthat are not labeled with an acceptor or donor for generating signals todetermine nucleotide sequence, may be modified by attaching anon-signal-producing adduct that has substantially the same effect ontranslocation speed as the signal-producing labels.

Nanopore Sequencing with Mutually Quenching Fluorescent Labels

The invention relates to the use of nanopores and fluorescent quenchingto sequentially identify monomers of polymer analytes. Such analysis ofpolymer analytes may be carried out on single polymer analytes or onpluralities of polymer analytes in parallel at the same time. In someembodiments, monomers are labeled with fluorescent labels that arecapable of at least three states while attached to a target polymer: (i)A quenched state wherein fluorescence of an attached fluorescent labelis quenched by a fluorescent label on an immediately adjacent monomer;for example, a fluorescent label attached to a polymer in accordancewith the invention is quenched when the labeled polymer is free in anaqueous solution. (ii) A sterically constrained state wherein a labeledpolymer is translocating through a nanopore such that the free-solutionmovements or alignments of an attached fluorescent label is disrupted orlimited so that there is little or no detectable signal generated fromthe fluorescent label. (iii) A transition state wherein a fluorescentlabel attached to a polymer transitions from the sterically constrainedstate to the quenched state as the fluorescent label exits the nanopore(during a “transition interval”) while the polymer translocates throughthe nanopore. In part, the invention is an application of the discoverythat during the transition interval a fluorescent label is capable ofgenerating a detectable fluorescent signal, Without the intention ofbeing limited by any theory underlying this discovery, it is believedthat the fluorescent signal generated during the transition interval isdue to a freely rotatable dipole. In both, the sterically constrainedstate as well as the quenched state the dipoles are limited in theirrotational freedom thereby reducing or limiting the number of emittedphotons. In some embodiments, the polymer is a polynucleotide, usually asingle stranded polynucleotide, such as, DNA or RNA, but especially DNA.In some embodiments, the invention includes a method for determining anucleotide sequence of a polynucleotide by recording signals generatedby attached fluorescent labels as they exit a nanopore one at a time asa polynucleotide translocates the nanopore. Upon exit, each attachedfluorescent label transitions during a transition interval from aconstrained state in the nanopore to a quenched state on thepolynucleotide in free solution. As mentioned above, during thistransition interval or period the fluorescent label is capable ofemitting a detectable fluorescent signal indicative of the nucleotide itis attached to.

In some embodiments, a nucleotide sequence of a target polynucleotide isdetermined by carrying out four separate reactions in which copies ofthe target polynucleotide have each of its four different kinds ofnucleotide (A, C, G and T) labeled with a single fluorescent label. In avariant of such embodiments, a nucleotide sequence of a targetpolynucleotide is determined by carrying out four separate reactions inwhich copies of the target polynucleotide have each of its fourdifferent kinds of nucleotide (A, C, G and T) labeled with onefluorescent label while at the same time the other nucleotides on thesame target polynucleotide are labeled with a second fluorescent label.For example, if a first fluorescent label is attached to A's of thetarget polynucleotide in a first reaction, then a second fluorescentlabel is attached to C's. G's and T's (i.e. to the “not-A” nucleotides)of the target polynucleotides in the first reaction. Likewise, incontinuance of the example, in a second reaction, the first label isattached to C's of the target polynucleotide and the second fluorescentlabel is attached to A's, G's and T's (i.e. to the “not-C” nucleotides)of the target polynucleotide. And so on, for nucleotides G and T.

The same labeling scheme may be expressed in terms of conventionalterminology for subsets of nucleotide types; thus, in the above example,in a first reaction, a first fluorescent label is attached to A's and asecond fluorescent label is attached to B's; in a second reaction, afirst fluorescent label is attached to C's and a second fluorescentlabel is attached to D's; in a third reaction, a first fluorescent labelis attached to G's and a second fluorescent label is attached to H's;and in a fourth reaction, a first fluorescent label is attached to T'sand a second fluorescent label is attached to V's.

In some embodiments, a feature of the invention is the labeling ofsubstantially all monomers of a polymer analytes with fluorescent dyesor labels that are members of a mutually quenching set. Such sets offluorescent dyes have the following properties: (i) each member quenchesfluorescence of every member (for example, by FRET or by static orcontact mechanisms), and (ii) each member generates a distinctfluorescent signal when excited and when in a non-quenching state. Thatis, if a mutually quenching set consists of two dyes, D1 and D2, then(i) D1 is self-quenched (e.g. by contact quenching with another D1molecule) and it is quenched by D2 (e.g. by contact quenching) and (ii)D2 is self-quenched (e.g. by contact quenching with another D2 molecule)and it is quenched by D1 (e.g. by contact quenching). Guidance forselecting fluorescent dyes or labels for mutually quenching sets may befound in the following references, which are incorporated herein byreference: Johansson, Methods in Molecular Biology, 335: 17-29 (2006);Marras et al, Nucleic Acids Research, 30: e122 (2002); and the like.Exemplary mutually quenching sets of fluorescent dyes, or labels, may beselected from rhodamine dyes, fluorescein dyes and cyanine dyes. In oneembodiment, a mutually quenching set may comprise the rhodamine dye,TAMRA, and the fluorescein dye, FAM. In another embodiment, mutuallyquenching sets of fluorescent dyes may be formed by selecting two ormore dyes from the group consisting of Oregon Green 488. Fluorescein-EX,fluorescein isothiocyanate, Rhodamine Red-X, Lissamine rhodamine B,Calcein, Fluorescein, Rhodamine, one or more BODIPY dyes, Texas Red,Oregon Green 514, and one or more Alexa Fluors. Representative BODIPYdyes include BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY 581/591, BODIPYTR, BODIPY 630/650 and BODIPY 650/665. Representative Alexa Fluorsinclude Alexa Fluor 350, 405, 430, 488, 500, 514, 532, 546, 555, 568,594, 610, 633, 635, 647, 660, 680, 700, 750 and 790.

In some embodiments, fluorescent labels are members of a FRET pair. AFRET pair generally is one or more FRET donors and one or more FRETacceptors where each donor is capable of a FRET reaction with eachacceptor. In one aspect, this means that the donors of the FRET pairhave an emission spectrum that substantially overlaps the absorptionspectrum of the acceptors. In another aspect, the transition dipole ofthe donor and the acceptor have to be aligned in a way that allowsefficient energy transfer. In some aspects, the invention in part isbased on the discovery and appreciation of a fluorescence, particularly,FRET suppressing property of nanopores and the application of thisproperty to enable detection of labeled polymers translocating through ananopore. It is believed, although the invention is not intended to belimited thereby, that a nanopore may be selected with a bore dimensionedso that a FRET pair label cannot orient to engage in a FRET interactionwhile translocating through the nanopore. The dipoles of the labels ofthe polynucleotide in the bore of the nanopore are constrained in theirrotational freedom based on the limited diameter of the nanopore. Thisreduction in dipole alignment with the alignment of the correspondingFRET pair attached to the nanopore limits the FRET efficiencydramatically. Labeled polynucleotides can engage in a FRET interactionafter exiting the nanopore at which point the FRET acceptor or donor onthe polymer (e.g. polynucleotide) regains rotational freedom whichallows for a FRET event.

A wide range of embodiments of the above may be implemented depending onthe type of analytes being detected, the types of donors and acceptorsemployed, the physical arrangement of the nanopores, donors andacceptors, whether analytes are labeled with donors or with acceptors,and the like. In one embodiment, analytes measured by the invention areacceptor-labeled polymers, especially acceptor-labeled polynucleotides.In one species of the latter embodiment, different nucleotides of apolynucleotide analyte are labeled with one or more different kinds ofacceptors, so that a nucleotide sequence of the polynucleotide may bedetermined from measuring FRET signals generated as it translocatesthrough a nanopore. In another embodiment, analytes measured by theinvention are donor-labeled polymers, especially donor-labeledpolynucleotides. The sequence of the polynucleotide may be determinedfrom measuring FRET signals as it translocates through a nanopore. Inyet another embodiment of the present invention, at least one of thefour nucleotides of a polynucleotide analyte is labeled with a member ofa FRET pair. The positions of the labeled nucleotides in thepolynucleotide are determined by translocating the labeledpolynucleotide through a labeled nanopore and measuring FRET events. Bylabeling the remaining nucleotides of the same polynucleotide sample andsubsequently translocating said samples through a labeled nanopore,sub-sequences of the polynucleotide are generated. Such sub-sequencescan be re-aligned resulting in a full sequence of the polynucleotide.

Some of the above aspects and embodiments of the invention areillustrated diagrammatically in FIG. 3. Polymer analyte (3000), such asa polynucleotide, is driven, e.g. electrophoretically, through nanopore(3002), which constrains the conformation of polymer (3000) so that itsmonomeric units translocate through the nanopore in the same order astheir primary sequence in the polymer. In the embodiment shown in FIG.3, fluorescent labels are assumed to be members of FRET pairs, but thisis not intended to limit the present invention; fluorescent labels mayalso include fluorescent labels that are directly excited, for examplewith a laser emitting at an appropriate wavelength, to generate afluorescent signal.

As mentioned above, whenever an acceptor-labeled monomeric unit iswithin the bore of nanopore (3002). FRET interactions between suchacceptors and the donors of its FRET pair are suppressed becauseacceptors are in a constrained state (3014). Such suppression typicallymeans that no detectable FRET signal is produced even if such acceptorsare within a FRET distance of a donor, for example, due to unfavorableorientation of the acceptor and donor dipoles. On the other hand, whenan acceptor-labeled monomeric unit emerges from the bore of, or exits,the nanopore into transition zone (3008). FRET interaction (3010) occursand FRET emission (3016) is produced and detected by detector (3018)until the acceptor enters a self-quenching state (3011) with an adjacentacceptor and as the distance between the acceptor and donor increaseswith the movement of polymer (3000) out of FRET interaction distance.Signal (3022) is produced by a single acceptor as it moves throughtransition zone (3008). Transition zone (3008), which is a spatialregion immediately adjacent to exit (3015) of nanopore (3002), isdefined by several factors, including the speed of the translocation ofpolymer (3000) through nanopore (3002), the vibrational and rotationalmobility of the fluorescent labels, the physiochemical nature of thefluorescent labels, and the like. In FIG. 3, only one type of monomericunit, illustrated as solid circles (3004) carries a first fluorescentlabel (designated as “a”); the rest of the monomeric units, illustratedas speckled circles (3006), carry a second fluorescent label (designatedas “b”). In this embodiment, first fluorescent labels quench adjacentfirst fluorescent labels and adjacent second fluorescent labels;likewise, second fluorescent labels quench adjacent first fluorescentlabels and adjacent second fluorescent labels; moreover, the first andsecond fluorescent labels generate FRET signals that are distinguishablefrom one another, for example, recorded signal (3022) for label “a” andrecorded signal (3023) for label “b” in FIG. 3, so that each fluorescentlabel (and hence, monomer) may be identified by a signal detected bydetector (3018).

In some embodiments, a nanopore is hybrid nanopore comprising a proteinnanopore inserted into a pore of a solid phase membrane, as describedabove. In hybrid nanopores, a first member of a FRET pair may beattached directly to the protein nanopore, or alternatively, directly tothe solid phase membrane using conventional linking chemistries, such as“click” chemistries, e.g. Kolb et al, Angew. Chem. Int. Ed., 4):2004-2021 (2001), or the like. In one embodiment, a first member of aFRET pair is attached directly or indirectly to the protein nanopore,for example, as discussed in reference to FIG. 2D. In anotherembodiment, the first member of the FRET pair is a donor and a quantumdot. Quantum dots are typically much larger than acceptors, especiallyacceptors that are organic dyes, which typically have molecular weightsin the range of from 200 to 2000 daltons.

In one embodiment, the present invention may be used in a method foranalyzing one or more polymer analytes, such as determining a nucleotidesequence of a polynucleotide, which comprises the following steps: (a)translocating polymer analytes through nanopores of a nanopore array,each nanopore having a bore and an exit, each polymer analyte comprisinga sequence of monomers, wherein substantially each monomer is labeledwith a fluorescent label such that fluorescent labels of adjacentmonomers are in a quenched state by self-quenching one another outsideof the nanopore and fluorescent labels are in a sterically constrainedstate and incapable of generating a detectable fluorescent signal insideof the nanopore; (b) exciting each florescent label at the exit of thenanopores as it transitions from a sterically constrained state to aquenched state so that a fluorescent signal is generated which isindicative of the monomer to which it is attached; (c) detecting thefluorescent signal to identify the monomer, wherein the nanopore arrayis an array of clusters. As used herein, “substantially every”,“substantially all”, or like terms, in reference to labeling monomers,particularly nucleotides, acknowledges that chemical labeling proceduresare rarely complete; to the extent practicable, the terms comprehendthat labeling reactions in connection with the invention are continuedto completion; in some embodiments, such completed labeling reactionsinclude labeling at least fifty percent of the monomers; in otherembodiments, such labeling reactions include labeling at least eightypercent of the monomers; in other embodiments, such labeling reactionsinclude labeling at least ninety-five percent of the monomers; in otherembodiments, such labeling reactions include labeling at leastninety-nine percent of the monomers.

In another embodiment the invention is directed to a device foranalyzing one or more labeled polymer analytes, such as a device fordetermining a nucleotide sequence of one or more labeled polynucleotideanalytes, such device comprising the following elements: (a) a solidphase membrane separating a first chamber and a second chamber, thesolid phase membrane having an array of nanopores each fluidlyconnecting the first chamber and the second chamber through a bore orlumen, the bore or lumen having a cross-sectional dimension such thatlabels of a labeled polymer translocating therethrough are stericallyconstrained so that detectable signals are not generated, and so thatthe labels of adjacent monomers of the labeled polymer areself-quenching; (b) an excitation source for exciting each label when itexits each nanopore and enters the second chamber so that a signal isgenerated indicative of a monomer to which the label is attached; and(c) a detector for collecting at least a portion of the signal generatedby each excited label; and (d) identifying the monomer to which theexcited label is attached by the collected signal whenever emitted froma sequence-able nanopore; and wherein the array of nanopores is an arrayof clusters of nanopores.

In another embodiment, the invention is directed to a system foranalyzing polymers comprising a polymer comprising monomers that aresubstantially all labeled with a mutually quenching dye set and ananopore device for sequentially detecting optical signals from the dyesof the mutually quenching dye set which are attached to the polymer.Such an embodiment for determining a sequence of a polynucleotide maycomprise the following elements: (a) a solid phase membrane separating afirst chamber and a second chamber, the solid phase membrane having anarray of apertures each connecting the first chamber and the secondchamber, and having a hydrophobic coating on at least one surface; (b) alipid layer disposed on the hydrophobic coating; (c) protein nanoporesimmobilized in the apertures, the protein nanopores each having a borewith an exit, and the protein nanopores interacting with the lipid layerto form a seal with the solid phase membrane in the apertures so thatfluid communication between the first chamber and the second chamberoccurs solely through the bore of the protein nanopore, and the proteinnanopores each being cross-sectionally dimensioned so that nucleotidesof the polynucleotide pass through the exit of the bore in sequence andso that fluorescent labels attached to the polynucleotide are stericallyconstrained; and (d) a first member of the FRET pair attached to thesolid phase membrane or the protein nanopore, so that whenevernucleotides of the polynucleotide emerge from the bore, a plurality ofthe nucleotides are within a FRET distance of the first member of theFRET pair; and wherein the array of apertures is an array of clusters ofapertures.

Arrays of Zero Mode Waveguides for Sequence Analysis

Arrays of zero mode waveguides have been developed for analyzing inparallel populations of single molecules each undergoing a sequence ofreactions that generate a corresponding sequence of optical signals,e.g. Levene et al, Science, 299: 682-686 (2003); Korlach et al, Proc.Natl. Acad. Sci., 105(4); 1176-1181 (2008). This approach has beenapplied to develop a high-throughput DNA sequencing instrument, Eid etal, Science, 323: 133-138 (2009). Such applications are furtherdisclosed in the following U.S. patents which are incorporated herein byreference: U.S. Pat. Nos. 7,302,146; 7,476,503; 7,906,284; 8,709,725;and the like. Typically, in arrays of zero mode waveguides used forthese applications, the waveguides are regularly spaced with aninter-waveguide distance sufficiently large that optical signals fromadjacent waveguides can be optically distinguished and do notsubstantially affect the values of collected signals. Such spacing isusually greater than the wavelength of the light comprising the opticalsignals, so that, as described above for nanopore arrays, the fullcapabilities of nano-engineering techniques for close placement offeatures is not employed. Accordingly, the efficiency of such methodsmay be improved by employing arrays of clusters in accordance with theinvention.

In some embodiments, an improved method of performing multiple chemicalreactions involving a plurality of reaction samples may be performedwith the following steps: (a) providing an array of nanowells; (b)placing the plurality of reaction samples comprising labeled reactantsinto the nanowells of the array, wherein a separate reaction sample isplaced into a different nanowell in the array; (c) subjecting the arrayto conditions suitable for formation of products of the chemicalreactions; and (d) detecting the formation of the products with anoptical system operationally associated therewith, and wherein nanowellsof the array are arranged in clusters such that each different clusterof nanowells is disposed within a different resolution limited area andsuch that an average number of nanowells in each cluster is greater thanzero. In some embodiments, nanowells of an array each comprise anoptical confinement and/or a zero mode waveguide, as described in U.S.Pat. No. 7,302,146.

In some embodiments, an improved method of sequencing a plurality oftarget nucleic acid molecules may be performed by the following steps:(a) providing an array of nanowells, wherein nanowells of the array arearranged in clusters such that each different cluster of nanowells isdisposed within a different resolution limited area and such that anaverage number of nanowells in each cluster is greater than zero, andwherein each nanowell provide an effective observation volume thatpermits observation of individual molecules; and an optical systemoperatively coupled to the nanowells that detects signals from theeffective observation volume of the nanowells; (b) mixing in thenanowells the plurality of target nucleic acid molecules, primerscomplementary to the target nucleic acid molecules, polymerizationenzymes, and more than one type of nucleotides or nucleotide analogs tobe incorporated into a plurality of nascent nucleotide strands, eachstrand being complementary to a respective target nucleic acid molecule;(c) subjecting the mixture of step (b) to a polymerization reactionunder conditions suitable for formation of the nascent nucleotidestrands by template-directed polymerization of the nucleotides ornucleotide analogs; (d) illuminating the nanowells with an incidentlight beam; and (c) identifying the nucleotides or the nucleotideanalogs incorporated into the each nascent nucleotide strand. As above,in some embodiments, nanowells of an array each comprise an opticalconfinement and/or a zero mode waveguide, as described in U.S. Pat. No.7,302,146.

Definitions

“Cluster” in reference to an array of nanostructures means adistribution of a plurality of groups or collections of nanostructureswherein each group occupies a separate area of the array and whereinintra-group nanostructure-to-nanostructure distances are much less thaninter-group nanostructure-to-nanostructure distances. In someembodiments, such a distribution is substantially planar; that is, ifnanostructures are spaced relative to a surface, the curvature of suchsurface is small in the proximity of a cluster. In some embodiments, acluster of nanostructures is encompassed by a resolution limited area,such that nanostructures of different clusters are in differentresolution limited areas. The number of nanostructures within a clustermay vary widely. In some embodiments, nanostructure arrays may befabricated with a predetermined number of nanostructures within eachcluster of the array. For example, clusters of an array may each have aplurality of nanostructures; in other embodiments, clusters of an arraymay each have from 1 to 100 nanostructures; in other embodiments,clusters may each have from 2 to 50 nanostructures; in otherembodiments, clusters may each have from 2 to 16 nanostructures. In someembodiments, each cluster of an array may have the same number ofnanostructures. In other embodiments, the number of nanostructureswithin a cluster may be a random variable, such that an average, orexpected, number, and possibly its variance, characterizes clusterswithin an array. In some embodiments, clusters of nanostructures areclusters of nanopores; in other embodiments, clusters of nanostructuresare clusters of nanowells, including, but not limited to, nanowells thatare zero mode waveguides. In some embodiments, for example, wherenanostructures comprise protein nanopores, a random variablerepresenting the number of nanopores in a cluster may be a Poissonrandom variable whose average value depends on the concentration ofprotein nanopores in a solution used to load an array of apertures.

“FRET” or “Förster, or fluorescence, resonant energy transfer” means anon-radiative dipole-dipole energy transfer mechanism from an exciteddonor fluorophore to an acceptor fluorophore in a ground state. The rateof energy transfer in a FRET interaction depends on the extent ofspectral overlap of the emission spectrum of the donor with theabsorption spectrum of the acceptor, the quantum yield of the donor, therelative orientation of the donor and acceptor transition dipoles, andthe distance between the donor and acceptor molecules, Lakowitz,Principles of Fluorescence Spectroscopy, Third Edition (Springer, 2006).FRET interactions of particular interest are those which result aportion of the energy being transferred to an acceptor, in turn, beingemitted by the acceptor as a photon, with a frequency lower than that ofthe light exciting its donor (i.e. a “FRET signal”). “FRET distance”means a distance between a FRET donor and a FRET acceptor over which aFRET interaction can take place and a detectable FRET signal produced bythe FRET acceptor.

“Kit” refers to any delivery system for delivering materials or reagentsfor carrying out a method of the invention. In the context of reactionassays, such delivery systems include systems that allow for thestorage, transport, or delivery of reaction reagents (e.g., fluorescentlabels, such as mutually quenching fluorescent labels, fluorescent labellinking agents, enzymes. etc. in the appropriate containers) and/orsupporting materials (e.g., buffers, written instructions for performingthe assay etc.) from one location to another. For example, kits includeone or more enclosures (e.g., boxes) containing the relevant reactionreagents and/or supporting materials. Such contents may be delivered tothe intended recipient together or separately. For example, a firstcontainer may contain an enzyme for use in an assay, while a second ormore containers contain mutually quenching fluorescent labels.

“Microfluidics” device or “nanofluidics” device, used interchangeablyherein, each means an integrated system for capturing, moving, mixing,dispensing or analyzing small volumes of fluid, including samples(which, in turn, may contain or comprise cellular or molecular analytesof interest), reagents, dilutants, buffers, or the like. Generally,reference to “microfluidics” and “nanofluidics” denotes different scalesin the size of devices and volumes of fluids handled. In someembodiments, features of a microfluidic device have cross-sectionaldimensions of less than a few hundred square micrometers and havepassages, or channels, with capillary dimensions, e.g. having maximalcross-sectional dimensions of from about 500 μm to about 0.1 μm. In someembodiments, microfluidics devices have volume capacities in the rangeof from 1 μL to a few nL, e.g. 10-100 nL. Dimensions of correspondingfeatures, or structures, in nanofluidics devices are typically from 1 to3 orders of magnitude less than those for microfluidics devices. Oneskilled in the art would know from the circumstances of a particularapplication which dimensionality would be pertinent. In someembodiments, microfluidic or nanofluidic devices have one or morechambers, ports, and channels that are interconnected and in fluidcommunication and that are designed for carrying out one or moreanalytical reactions or processes, either alone or in cooperation withan appliance or instrument that provides support functions, such assample introduction, fluid and/or reagent driving means, such aspositive or negative pressure, acoustical energy, or the like,temperature control, detection systems, data collection and/orintegration systems, and the like. In some embodiments, microfluidicsand nanofluidics devices may further include valves, pumps, filters andspecialized functional coatings on interior walls, e.g. to preventadsorption of sample components or reactants, facilitate reagentmovement by electroosmosis, or the like. Such devices may be fabricatedin or as a solid substrate, which may be glass, plastic, or other solidpolymeric materials, and may have a planar format for ease of detectingand monitoring sample and reagent movement, especially via optical orelectrochemical methods. In some embodiments, such devices aredisposable after a single use. In some embodiments, microfluidic andnanofluidic devices include devices that form and control the movement,mixing, dispensing and analysis of droplets, such as, aqueous dropletsimmersed in an immiscible fluid, such as a light oil. The fabricationand operation of microfluidics and nanofluidics devices are well-knownin the art as exemplified by the following references that areincorporated by reference: Ramsey. U.S. Pat. Nos. 6,001,229; 5,858,195;6,010,607; and 6,033,546; Soane et al, U.S. Pat. Nos. 5,126,022 and6,054,034; Nelson et al, U.S. Pat. No. 6,613,525; Maher et al, U.S. Pat.No. 6,399,952; Ricco et al, International patent publication WO02/24322; Bjornson et al, International patent publication WO 99/19717;Wilding et al, U.S. Pat. Nos. 5,587,128; 5,498,392; Sia et al,Electrophoresis, 24: 3563-3576 (2003); Unger et al, Science, 288:113-116 (2000); Enzelberger et al, U.S. Pat. No. 6,960,437; Cao,“Nanostructures & Nanomaterials: Synthesis, Properties & Applications,”(Imperial College Press, London, 2004); Haeberle et al, LabChip, 7:1094-1110 (2007); Cheng et al, Biochip Technology (CRC Press, 2001); andthe like.

“Nanopore” means any opening positioned in a substrate that allows thepassage of analytes through the substrate in a predetermined ordiscernable order, or in the case of polymer analytes, passage of theirmonomeric units through the substrate in a predetermined or discernibleorder. In the latter case, a predetermined or discernible order may bethe primary sequence of monomeric units in the polymer. Examples ofnanopores include proteinaceous or protein based nanopores, synthetic orsolid state nanopores, and hybrid nanopores comprising a solid statenanopore having a protein nanopore embedded therein. A nanopore may havean inner diameter of 1-10 nm or 1-5 nm or 1-3 nm. Examples of proteinnanopores include but are not limited to, alpha-hemolysin,voltage-dependent mitochondrial porin (VDAC), OmpF, OmpC, MspA and LamB(maltoporin), e.g. disclosed in Rhee, M. et al., Trends inBiotechnology, 25(4) (2007): 174-181; Bayley et al (cited above);Gundlach et al, U.S. patent publication 2012/0055792; and the like,which are incorporated herein by reference. Any protein pore that allowsthe translocation of single nucleic acid molecules may be employed. Ananopore protein may be labeled at a specific site on the exterior ofthe pore, or at a specific site on the exterior of one or more monomerunits making up the pore forming protein. Pore proteins are chosen froma group of proteins such as, but not limited to, alpha-hemolysin, MspA,voltage-dependent mitochondrial porin (VDAC). Anthrax porin, OmpF, OmpCand LamB (maltoporin). Integration of the pore protein into the solidstate hole is accomplished by attaching a charged polymer to the poreprotein. After applying an electric field the charged complex iselectrophoretically pulled into the solid state hole. A syntheticnanopore, or solid-state nanopore, may be created in various forms ofsolid substrates, examples of which include but are not limited tosilicones (e.g. Si3N4, SiO2), metals, metal oxides (e.g. Al2O3)plastics, glass, semiconductor material, and combinations thereof. Asynthetic nanopore may be more stable than a biological protein porepositioned in a lipid bilayer membrane. A synthetic nanopore may also becreated by using a carbon nanotube embedded in a suitable substrate suchas but not limited to polymerized epoxy. Carbon nanotubes can haveuniform and well-defined chemical and structural properties. Varioussized carbon nanotubes can be obtained, ranging from one to hundreds ofnanometers. The surface charge of a carbon nanotube is known to be aboutzero, and as a result, electrophoretic transport of a nucleic acidthrough the nanopore becomes simple and predictable (Ito, T. et al.,Chem. Commun. 12 (2003): 1482-83). The substrate surface of a syntheticnanopore may be chemically modified to allow for covalent attachment ofthe protein pore or to render the surface properties suitable foroptical nanopore sequencing. Such surface modifications can be covalentor non-covalent. Most covalent modification include an organosilanedeposition for which the most common protocols are described: 1)Deposition from aqueous alcohol. This is the most facile method forpreparing silylated surfaces. A 95% ethanol-5% water solution isadjusted to pH 4.5-5.5 with acetic acid. Silane is added with stirringto yield a 2% final concentration. After hydrolysis and silanol groupformation the substrate is added for 2-5 min. After rinsed free ofexcess materials by dipping briefly in ethanol. Cure of the silane layeris for 5-10 min at 110 degrees Celsius. 2) Vapor Phase Deposition.Silanes can be applied to substrates under dry aprotic conditions bychemical vapor deposition methods. These methods favor monolayerdeposition. In closed chamber designs, substrates are heated tosufficient temperature to achieve 5 mm vapor pressure. Alternatively,vacuum can be applied until silane evaporation is observed. 3) Spin-ondeposition. Spin-on applications can be made under hydrolytic conditionswhich favor maximum functionalization and polylayer deposition or dryconditions which favor monolayer deposition. In some embodiments, singlenanopores are employed with methods of the invention. In otherembodiments, a plurality of nanopores are employed. In some of thelatter embodiments, a plurality of nanopores is employed as an array ofnanopores, usually disposed in a planar substrate, such as a solid phasemembrane. Nanopores of a nanopore array may be spaced regularly, forexample, in a rectilinear pattern, or may be spaced randomly. In apreferred embodiment, nanopores are spaced regularly in a rectilinearpattern in a planar solid phase substrate.

“Nanostructure” (used interchangeably with “nanoscale structure” and“nanoscale feature”) means a structure that has at least one dimensionwithin a range of a few nanometers to several hundred nanometers, forexample, from 1 to 1000 nanometers. In some applications, such range isfrom 2 to 500 nanometers; in other applications, such range is from 3 to500 nanometers. The shape and geometry of nanostructures may vary widelyand include, but are not limited to, nanopores, nanowells,nanoparticles, and any other convenient shapes particularly suitable forcarrying out sequences of reactions. In some embodiments, nanostructuresmay be protein nanopores operationally associated with a solid phasemembrane. Some nanostructures, such as, nanopores and nanowells, may beformed in a larger common substrate, such as a solid phase membrane, orother solid, to form arrays of nanopores or nanowells. Nanostructures ofparticular interest are those capable of supporting or containing achemical, physical (e.g. FRET), enzymatic and/or binding reaction or asequence of such reactions. In some embodiments, a nanostructure, suchas a nanowell, encloses a volume that is less than one nanoliter (10×−9liter), less than one picoliter, or less than one femtoliter. In otherembodiments, each of the individual nanowells provides a volume that isless than 1000 zeptoliters, 100 zeptoliters, 80 zeptoliters, or lessthan 50 zeptoliters, or less than 1 zeptoliter, or even less than 100yactoliters. In some embodiments, nanowells comprise zero modewaveguides.

“Peptide,” “peptide fragment,” “polypeptide,” “oligopeptide,” or“fragment” in reference to a peptide are used synonymously herein andrefer to a compound made up of a single unbranched chain of amino acidresidues linked by peptide bonds. Amino acids in a peptide orpolypeptide may be derivatized with various moieties, including but notlimited to, polyethylene glycol, dyes, biotin, haptens, or likemoieties. The number of amino acid residues in a protein or polypeptideor peptide may vary widely; however, in some embodiments, protein orpolypeptides or peptides referred to herein may have 2 from to 70 aminoacid residues; and in other embodiments, they may have from 2 to 50amino acid residues. In other embodiments, proteins or polypeptides orpeptides referred to herein may have from a few tens of amino acidresidues, e.g. 20, to up to a thousand or more amino acid residues, e.g.1200). In still other embodiments, proteins, polypeptides, peptides, orfragments thereof, may have from 10 to 1000 amino acid residues; or theymay have from 20 to 500 amino acid residues; or they may have from 20 to200 amino acid residues.

“Polymer” means a plurality of monomers connected into a linear chain.Usually, polymers comprise more than one type of monomer, for example,as a polynucleotide comprising A's, C's, G's and T's, or a polypeptidecomprising more than one kind of amino acid. Monomers may includewithout limitation nucleosides and derivatives or analogs thereof andamino acids and derivatives and analogs thereof. In some embodiments,polymers are polynucleotides, whereby nucleoside monomers are connectedby phosphodiester linkages, or analogs thereof.

“Polynucleotide” or “oligonucleotide” are used interchangeably and eachmean a linear polymer of nucleotide monomers. Monomers making uppolynucleotides and oligonucleotides are capable of specifically bindingto a natural polynucleotide by way of a regular pattern ofmonomer-to-monomer interactions, such as Watson-Crick type of basepairing, base stacking. Hoogsteen or reverse Hoogsteen types of basepairing, or the like. Such monomers and their internucleosidic linkagesmay be naturally occurring or may be analogs thereof. e.g. naturallyoccurring or non-naturally occurring analogs. Non-naturally occurringanalogs may include PNAs, phosphorothioate internucleosidic linkages,bases containing linking groups permitting the attachment of labels,such as fluorophores, or haptens, and the like. Whenever the use of anoligonucleotide or polynucleotide requires enzymatic processing, such asextension by a polymerase, ligation by a ligase, or the like, one ofordinary skill would understand that oligonucleotides or polynucleotidesin those instances would not contain certain analogs of internucleosidiclinkages, sugar moieties, or bases at any or some positions.Polynucleotides typically range in size from a few monomeric units, e.g.5-40, when they are usually referred to as “oligonucleotides,” toseveral thousand monomeric units. Whenever a polynucleotide oroligonucleotide is represented by a sequence of letters (upper or lowercase), such as “ATGCCTG,” it will be understood that the nucleotides arein 5′→3′ order from left to right and that “A” denotes deoxyadenosine.“C” denotes deoxycytidine. “G” denotes deoxyguanosine, and “T” denotesthymidine, “I” denotes deoxyinosine, “U” denotes uridine, unlessotherwise indicated or obvious from context. Unless otherwise noted theterminology and atom numbering conventions will follow those disclosedin Strachan and Read, Human Molecular Genetics 2 (Wiley-Liss, New York,1999). Usually polynucleotides comprise the four natural nucleosides(e.g. deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine forDNA or their ribose counterparts for RNA) linked by phosphodiesterlinkages; however, they may also comprise non-natural nucleotideanalogs, e.g. including modified bases, sugars, or internucleosidiclinkages. It is clear to those skilled in the art that where an enzymehas specific oligonucleotide or polynucleotide substrate requirementsfor activity, e.g. single stranded DNA, RNA/DNA duplex, or the like,then selection of appropriate composition for the oligonucleotide orpolynucleotide substrates is well within the knowledge of one ofordinary skill, especially with guidance from treatises, such asSambrook et al, Molecular Cloning, Second Edition (Cold Spring HarborLaboratory, New York, 1989), and like references. Likewise, theoligonucleotide and polynucleotide may refer to either a single strandedform or a double stranded form (i.e. duplexes of an oligonucleotide orpolynucleotide and its respective complement). It will be clear to oneof ordinary skill which form or whether both forms are intended from thecontext of the terms usage.

“Sequence determination”, “sequencing” or “determining a nucleotidesequence” or like terms in reference to polynucleotides includesdetermination of partial as well as full sequence information of thepolynucleotide. That is, the terms include sequences of subsets of thefull set of four natural nucleotides, A, C, G and T, such as, forexample, a sequence of just A's and C's of a target polynucleotide. Thatis, the terms include the determination of the identities, ordering, andlocations of one, two, three or all of the four types of nucleotideswithin a target polynucleotide. In some embodiments, the terms includethe determination of the identities, ordering, and locations of two,three or all of the four types of nucleotides within a targetpolynucleotide. In some embodiments sequence determination may beaccomplished by identifying the ordering and locations of a single typeof nucleotide, e.g. cytosines, within the target polynucleotide “catcgc. . . ” so that its sequence is represented as a binary code, e.g.“100101 . . . “representing” c-(not c)(not c)c-(not c)-c . . . ” and thelike. In some embodiments, the terms may also include subsequences of atarget polynucleotide that serve as a fingerprint for the targetpolynucleotide; that is, subsequences that uniquely identify a targetpolynucleotide, or a class of target polynucleotides, within a set ofpolynucleotides, e.g. all different RNA sequences expressed by a cell.

This disclosure is not intended to be limited to the scope of theparticular forms set forth, but is intended to cover alternatives,modifications, and equivalents of the variations described herein.Further, the scope of the disclosure fully encompasses other variationsthat may become obvious to those skilled in the art in view of thisdisclosure. The scope of the present invention is limited only by theappended claims.

What is claimed is:
 1. A device for analyzing polymers each havingoptical labels attached to a sequence of monomers, the devicecomprising: a nanopore array separating a first chamber and a secondchamber, wherein nanopores of the nanopore array each connect the firstchamber and the second chamber and are arranged in clusters such thateach different cluster of nanopores is disposed within a differentresolution limited area; a polymer translocating system for movingpolymers in the first chamber to the second chamber through thenanopores of the nanopore array; and a detection system for collectingoptical signals generated by optical labels attached to polymerswhenever an optical label exits a nanopore within a resolution limitedarea.
 2. The device of claim 1 wherein said clusters are arranged in arectilinear array or in a hexagonal array.
 3. The device of claim 1wherein each of said clusters comprises a plurality of nanopores or eachof said clusters comprises a number of nanopores which is a randomvariable with an average value greater than zero.
 4. The device of claim1 wherein said polymers are polynucleotides and wherein said detectionsystem determines numbers of functional nanopores within each of saidresolution limited areas from said collected optical signals and variesan electrical field across said nanopore array to maximize a rate ofsequence determination by said nanopore array.
 5. A method of analyzingpolymers each having a sequence of optical labels comprising: providinga nanopore array wherein each nanopore has a bore capable of providingfluid communication between a first chamber and a second chamber, andthe nanopore array having at least one resolution limited areacontaining a plurality of nanopores; translocating polymers throughnanopores of the nanopore array; collecting and integrating opticalsignals from polymers translocating through nanopores in each of theresolution limited areas during a predetermined interval to obtain anintegrated signal for each of the resolution limited areas; anddetermining a number of functional nanopores within each of theresolution limited areas by a relative level of its integrated signal.6. The method of claim 5 further including the step of selecting apolymer concentration and/or polymer flux to maximize sequencingthroughput of the nanopore array.
 7. The method of claim 6 wherein saidpolymers are polynucleotides and said step of selecting comprisesadjusting said polymer flux to maximize sequencing throughput by varyingan electrical field strength across said nanopore array.
 8. A device foranalyzing polymers each having a sequence of optical labels comprising:a solid phase membrane separating a first chamber and a second chamber,the solid phase membrane having an array of apertures each connectingthe first chamber and the second chamber, the apertures of the arraybeing arranged in clusters each having a plurality of apertures and eachdifferent cluster being disposed within a different resolution limitedarea; protein nanopores immobilized in the apertures, the immobilizedprotein nanopores having an active fraction wherein each immobilizedprotein of the active fraction is capable of translocating a polymerfrom the first chamber to the second chamber through a bore; and adetection system for collecting optical signals generated by opticallabels whenever an optical label exits a bore of a protein nanoporewithin a resolution limited area.
 9. The device of claim 8 wherein saidpolymers comprise polynucleotides and wherein said detection systemdetermines numbers of functional nanopores within each of saidresolution limited areas from said collected optical signals and variesan electrical field across said nanopore array to maximize a rate ofdetermining said sequences of optical labels by said nanopore array. 10.A method of sequencing polynucleotides each having a sequence of opticallabels comprising: (a) translocating single stranded polynucleotides ata concentration and flux through a nanopore array, wherein substantiallyevery nucleotide of each single stranded polynucleotides is labeled withan optical label capable of generating an optical signal indicative ofthe nucleotide to which it is attached, and wherein the nanopore arraycomprises clusters of nanopores, each cluster comprising a number ofnanopores, wherein each different cluster is disposed within a differentresolution limited area and wherein the number of nanopores in eachcluster is either greater than one or is a random variable with anaverage value greater than zero; (b) exposing the optical labels of eachnucleotide to excitation radiation upon exiting a nanopore; (c)measuring in each resolution limited region on the nanopore arrayoptical signals generated by optical labels exiting nanopores toidentify the nucleotide to which the optical label is attached wheneversuch optical signals are from a single optical label; and (d)determining a nucleotide sequence of the polynucleotide from a sequenceof optical signals from single optical labels.
 11. The method of claim10 wherein concentration of said polynucleotides and/or flux of saidpolynucleotides through said nanopore array are adjusted to maximizesequence-able nanopores in the array.
 12. The method of claim 10 whereinsaid flux of said single stranded polynucleotides is controlled tomaximize the number of sequence-able nanopores in the array bycontrolling an electrical potential across said nanopore array.
 13. Anapparatus for detecting sequences of optical signals from parallelreactions on an array, the apparatus comprising: an array of nanoporeseach comprising a reaction site and each capable of confining a reactionthat generates a sequence of optical signals, the nanopores of the arraybeing arranged in clusters each comprising a number of nanopores andeach different cluster of nanopores being disposed within a differentresolution limited area; and an optical system operatively associatedwith the array for detecting optical signals from the reactions.
 14. Theapparatus of claim 13 wherein said number of nanopores in each of saidclusters is either greater than one or a random variable with an averagevalue greater than zero.
 15. The apparatus of claim 14 wherein saidarray comprises a solid phase membrane separating a first chamber and asecond chamber, the solid phase membrane having a plurality of apertureseach connecting the first chamber and the second chamber and each beingdisposed in a cluster; and protein nanopores immobilized in theapertures, the immobilized protein nanopores having an active fractionwherein each immobilized protein of the active fraction is capable oftranslocating a polymer from the first chamber to the second chamberthrough a bore.
 16. The apparatus of claim 15 wherein said reactions atsaid protein nanopores are FRET interactions between donors andacceptors of a FRET pair, each acceptor being attached to a monomer ofsaid polymer and each acceptor being capable of generating an opticalsignal indicative of the monomer to which it is attached.
 17. Theapparatus of claim 14 wherein said array comprises a solid phasemembrane separating a first chamber and a second chamber, the solidphase membrane having a plurality of apertures each connecting the firstchamber and the second chamber and each being spanned by a lipidbilayer; the lipid bilayers having disposed therein protein nanopores toform said clusters, such that said number of protein nanopores in eachcluster is said random variable with said average value greater thanzero.
 18. The apparatus of claim 17 wherein said reactions at saidprotein nanopores are FRET interactions between donors and acceptors ofa FRET pair, each donor being attached to a protein nanopore and eachacceptor being attached to a monomer of said polymer, wherein eachacceptor being capable of generating an optical signal indicative of themonomer to which it is attached.
 19. The apparatus of claim 14 whereinsaid nanopores include a nanowell comprising a zero mode waveguide.